Histone deacetylase inhibitors and compositions and methods of use thereof

ABSTRACT

Provided are certain histone deacetylase (HDAC) inhibitors of Formula I, compositions thereof, and methods of their use.

This application claims the benefit of priority of U.S. Provisionalapplication No. 61/785,759, filed Mar. 14, 2013, which is incorporatedherein by reference in its entirety.

Provided herein are certain histone deacetylase (HDAC) inhibitorycompounds, compositions thereof, and methods of their use.

Histone deacetylases (HDACs) are zinc-containing enzymes which catalysethe removal of acetyl groups from the ε-amino termini of lysine residuesclustered near the amino terminus of nucleosomal histones. There are 11known metal-dependent human histone deacetylases, grouped into fourclasses based on the structure of their accessory domains. Class Iincludes HDAC1, HDAC2, HDAC3, and HDAC8 and have homology to yeast RPD3.HDAC4, HDAC5, HDAC7, and HDAC9 belong to Class IIa and have homology toyeast HDAC1. HDAC6 and HDAC10 contain two catalytic sites and areclassified as Class IIb, whereas HDAC11 has conserved residues in itscatalytic center that are shared by both Class I and Class IIdeacetylases and is sometimes placed in Class IV.

Provided is a compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein

-   -   R is pyrimidine substituted with 1 or 2 groups independently        chosen from halo, C₁-C₄ alkyl, and C₁-C₄ haloalkyl;    -   each R¹ is independently chosen from halo, C₁-C₄ alkyl, and        C₁-C₄ haloalkyl; and    -   m is 1, 2 or 3.

Also provided is a pharmaceutical composition comprising a compound, ora pharmaceutically acceptable salt thereof, described herein and atleast one pharmaceutically acceptable excipient. Also provided is amethod of preparing a pharmaceutical composition comprising a compound,or a pharmaceutically acceptable salt thereof, described herein and atleast one pharmaceutically acceptable excipient.

Also provided is a method of treating a condition or disorder mediatedby at least one histone deacetylase in a subject in need of such atreatment which method comprises administering to the subject atherapeutically effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, described herein.

As used in the present specification, the following words, phrases andsymbols are generally intended to have the meanings as set forth below,except to the extent that the context in which they are used indicatesotherwise.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —CONH₂ isattached through the carbon atom.

“C₁-C₄ Alkyl” encompasses straight chain and branched chain having 1 to4 carbon atoms. Examples of C₁-C₄ alkyl groups include methyl, ethyl,propyl, isopropyl, n-butyl, sec-butyl, tert-butyl.

The term “halo” includes fluoro, chloro, bromo, and iodo, and the term“halogen” includes fluorine, chlorine, bromine, and iodine.

The term “C₁-C₄ haloalkyl” refers to a C₁-C₄ alkyl wherein 1 to 5hydrogen atoms are substituted with halo. Non-limiting examples of C₁-C₄haloalkyl groups include —CF₃, —CHF₂, —CFH₂, and —CH₂CF₃.

Compounds described herein include, but are not limited to, theiroptical isomers, racemates, and other mixtures thereof. In thosesituations, the single enantiomers or diastereomers, i.e., opticallyactive forms, can be obtained by asymmetric synthesis or by resolutionof the racemates. Resolution of the racemates can be accomplished, forexample, by conventional methods such as crystallization in the presenceof a resolving agent, or chromatography, using, for example a chiralhigh-pressure liquid chromatography (HPLC) column. In addition, suchcompounds include Z- and E-forms (or cis- and trans-forms) of compoundswith carbon-carbon double bonds. Where compounds described herein existin various tautomeric forms, the term “compound” is intended to includeall tautomeric forms of the compound. Such compounds also includecrystal forms including polymorphs and clathrates. Similarly, the term“salt” is intended to include all tautomeric forms and crystal forms ofthe compound.

“Pharmaceutically acceptable salts” include, but are not limited tosalts with inorganic acids, such as hydrochloride, phosphate,diphosphate, hydrobromide, sulfate, sulfinate, nitrate, and like salts;as well as salts with an organic acid, such as malate, maleate,fumarate, tartrate, succinate, citrate, acetate, lactate,methanesulfonate, p-toluenesulfonate, 2-hydroxyethylsulfonate, benzoate,salicylate, stearate, and alkanoate such as acetate, HOOC—(CH₂)_(n)—COOHwhere n is 0-4, and like salts. Similarly, pharmaceutically acceptablecations include, but are not limited to sodium, potassium, calcium,aluminum, lithium, and ammonium.

In addition, if the compounds described herein are obtained as an acidaddition salt, the free base can be obtained by basifying a solution ofthe acid salt. Conversely, if the product is a free base, an additionsalt, particularly a pharmaceutically acceptable addition salt, may beproduced by dissolving the free base in a suitable organic solvent andtreating the solution with an acid, in accordance with conventionalprocedures for preparing acid addition salts from base compounds. Thoseskilled in the art will recognize various synthetic methodologies thatmay be used to prepare non-toxic pharmaceutically acceptable additionsalts.

As used herein the terms “group”, “radical” or “fragment” are synonymousand are intended to indicate functional groups or fragments of moleculesattachable to a bond or other fragments of molecules.

The term “active agent” is used to indicate a compound or apharmaceutically acceptable salt thereof which has biological activity.In some embodiments, an “active agent” is a compound or pharmaceuticallyacceptable salt thereof having pharmaceutical utility. For example anactive agent may be an anti-neurodegenerative therapeutic.

The term “therapeutically effective amount” means an amount effective,when administered to a human or non-human patient, to provide atherapeutic benefit such as amelioration of symptoms, slowing of diseaseprogression, or prevention of disease e.g., a therapeutically effectiveamount may be an amount sufficient to decrease the symptoms of a diseaseresponsive to inhibition of HDAC activity.

As used herein, the terms “histone deacetylase” and “HDAC” are intendedto refer to anyone of a family of enzymes that remove N^(ε)-acetylgroups from the ε-amino groups of lysine residues of a protein (forexample, a histone, or tubulin). Unless otherwise indicated by context,the term “histone” is meant to refer to any histone protein, includingH1, H2A, H2B, H3, H4, and H5, from any species. In some embodiments, thehistone deacetylase is a human HDAC, including, but not limited to,HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-9, and HDAC-10. In someembodiments, the at least one histone deacetylase is selected fromHDAC-4, HDAC-5, HDAC-7, and HDAC-9. In some embodiments, the histonedeacetylase is a class IIa HDAC. In some embodiments, the histonedeacetylase is HDAC-4. In some embodiments, the histone deacetylase isHDAC-5. In some embodiments, the histone deacetylase is derived from aprotozoal or fungal source.

The terms “histone deacetylase inhibitor” and “inhibitor of histonedeacetylase” are intended to mean a compound, or a pharmaceuticallyacceptable salt thereof, described herein which is capable ofinteracting with a histone deacetylase and inhibiting its enzymaticactivity.

The term “a condition or disorder mediated by HDAC” or “a condition ordisorder mediated by histone deacetylase” as used herein refers to acondition or disorder in which HDAC and/or the action of HDAC isimportant or necessary, e.g., for the onset, progress, expression, etc.of that condition, or a condition which is known to be treated by HDACinhibitors (such as, e.g., trichostatin A).

The term “effect” describes a change or an absence of a change in cellphenotype or cell proliferation. “Effect” can also describe a change oran absence of a change in the catalytic activity of HDAC. “Effect” canalso describe a change or an absence of a change in an interactionbetween HDAC and a natural binding partner.

The term “inhibiting histone deacetylase enzymatic activity” is intendedto mean reducing the ability of a histone deacetylase to remove anacetyl group from a protein, such as but not limited to a histone ortubulin. The concentration of inhibitor which reduces the activity of ahistone deacetylase to 50% of that of the uninhibited enzyme isdetermined as the IC₅₀ value. In some embodiments, such reduction ofhistone deacetylase activity is at least 50%, such as at least about75%, for example, at least about 90%. In some embodiments, histonedeacetylase activity is reduced by at least 95%, such as by at least99%. In some embodiments, the compounds and pharmaceutical acceptablesalts thereof described herein have an IC₅₀ value less than 100nanomolar. In some embodiments, the compounds and pharmaceuticalacceptable salts thereof described herein have an IC₅₀ value from 100nanomolar to 1 micromolar. In some embodiments, the compounds andpharmaceutical acceptable salts thereof described herein have an IC₅₀value from 1 to 25 micromolar.

In some embodiments, such inhibition is specific, i.e., the histonedeacetylase inhibitor reduces the ability of a histone deacetylase toremove an acetyl group from a protein at a concentration that is lowerthan the concentration of the inhibitor that is required to produceanother, unrelated biological effect. In some embodiments, theconcentration of the inhibitor required for histone deacetylaseinhibitory activity is at least 2-fold lower, such as at least 5-foldlower, for example, at least 10-fold lower, such as at least 20-foldlower than the concentration required to produce an unrelated biologicaleffect.

“Treatment” or “treating” means any treatment of a disease state in apatient, including

-   -   a) preventing the disease, that is, causing the clinical        symptoms of the disease not to develop;    -   b) inhibiting the disease;    -   c) slowing or arresting the development of clinical symptoms;        and/or    -   d) relieving the disease, that is, causing the regression of        clinical symptoms.

“Subject” or “patient’ refers to an animal, such as a mammal, that hasbeen or will be the object of treatment, observation or experiment. Themethods described herein may be useful in both human therapy andveterinary applications. In some embodiments, the subject is a mammal;and in some embodiments the subject is human.

It is appreciated that certain features described herein, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures described herein, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables containedwithin Formula I, are specifically embraced by herein just as if eachand every combination was individually and explicitly recited, to theextent that such combinations embrace compounds that result in stablecompounds (i.e., compounds that can be isolated, characterized andtested for biological activity). In addition, all subcombinations of thechemical groups listed in the embodiments describing such variables, aswell as all subcombinations of uses and medical indications describedherein, such as those conditions or disorders mediated by HDAC, are alsospecifically embraced herein just as if each and every subcombination ofchemical groups and subcombination of uses and medical indications wasindividually and explicitly recited herein. In addition, someembodiments include every combination of one or more additional agentsdisclosed herein just as if each and every combination was individuallyand explicitly recited.

BRIEF DESCRIPTION OF THE DRAWINGS

Provided is a compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein

-   -   R is pyrimidine substituted with 1 or 2 groups independently        chosen from halo, C₁-C₄ alkyl, and C₁-C₄ haloalkyl;    -   each R¹ is independently chosen from halo, C₁-C₄ alkyl, and        C₁-C₄ haloalkyl; and    -   m is 1, 2 or 3.

In some embodiments, R is

wherein

-   -   each R² is independently chosen from halo, C₁-C₄ alkyl, and        C₁-C₄ haloalkyl;    -   n is 1 or 2; and    -   represents the point of connection to the rest of the molecule.

In some embodiments, R is

In some embodiments, each R² is independently C₁-C₄ alkyl. In someembodiments, each R² is C₁-C₄ haloalkyl.

In some embodiments, each R² is independently methyl or trifluoromethyl.

In some embodiments, n is 1.

In some embodiments, n is 1, and R² is methyl. In some embodiments, n is1, and R² is trifluoromethyl.

In some embodiments, R is

In some embodiments, m is 1.

In some embodiments, at least one R¹ is halo.

In some embodiments, at least one R¹ is fluoro.

In some embodiments, m is 1, and R¹ is 2-fluoro.

Also provided is a compound which is chosen from2-(2-fluorophenyl)-N-hydroxy-2-(4-(2-methylpyrimidin-5-yl)phenyl)acetamideand2-(2-fluorophenyl)-N-hydroxy-2-(4-(5-(trifluoromethyl)pyrimidin-2-yl)phenyl)acetamide,or a pharmaceutically acceptable salt thereof.

Methods for obtaining the compounds, or pharmaceutically acceptablesalts thereof, described herein will be apparent to those of ordinaryskill in the art, suitable procedures being described, for example, inexamples below, and in the references cited herein.

Also provided is a method for inhibiting at least one histonedeacetylase. Also provided is a use of at least one compound, orpharmaceutically acceptable salt thereof, described herein in themanufacture of medicament for inhibiting at least one histonedeacetylase. Also provided is at least one compound, or pharmaceuticallyacceptable salt thereof, described herein for use in a method forinhibiting at least one histone deacetylase. In some embodiments, the atleast one histone deacetylase is a Class IIa HDAC. In some embodiments,the at least one histone deacetylase has homology to yeast HDA1. In someembodiments, the at least one histone deacetylase is selected fromHDAC-4, HDAC-5, HDAC-7, and HDAC-9. In some embodiments, the inhibitionis in a cell. In some embodiments, the compound, or pharmaceuticallyacceptable salt thereof, described herein is selective for inhibiting atleast one class II histone deacetylase. In some embodiments, thecompound, or pharmaceutically acceptable salt thereof, described hereinis a selective inhibitor of HDAC-4 and/or HDAC-5.

Also provided is a method of treating a condition or disorder mediatedby HDAC in a subject in need of such a treatment, comprisingadministering to the subject a therapeutically effective amount of atleast one compound, or pharmaceutically acceptable salt thereof,described herein. Also provided is a use of at least one compound, orpharmaceutically acceptable salt thereof, described herein in themanufacture of medicament for the treatment of a condition or disordermediated by HDAC. Also provided is at least one compound, orpharmaceutically acceptable salt thereof, described herein for use in amethod for the treatment of the human or animal body by therapy. Alsoprovided is at least one compound, or pharmaceutically acceptable saltthereof, described herein for use in a method for the treatment of acondition or disorder.

In some embodiments, the condition or disorder mediated by HDACcomprises a neurodegenerative pathology. Accordingly, also provided is amethod of treating a neurodegenerative pathology mediated by HDAC in asubject in need of such a treatment, comprising administering to thesubject a therapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein.

In some embodiments, the neurodegenerative pathology is chosen fromAlzheimer's disease, Parkinson's disease, neuronal intranuclearinclusion disease (NIID), dentatorubral pallidolusyian atrophy (DRPLA),Friedreich's ataxia, Rubenstein-Taubi syndrome, and polyglutaminediseases such as Huntington's disease; spinocerebellar ataxia 1 (SCA 1),spinocerebellar ataxia 7 (SCA 7), seizures, striatonigral degeneration,progressive supranuclear palsy, torsion dystonia, spasmodic torticollis,dyskinesis, familial tremor, Gilles de la Tourette syndrome, diffuseLewy body disease, progressive supranuclear palsy, Pick's disease,primary lateral sclerosis, progressive neural muscular atrophy, spinalmuscular atrophy, hypertrophic interstitial polyneuropathy, retinitispigmentosa, hereditary optic atrophy, hereditary spastic paraplegia,Shy-Drager syndrome, Kennedy's disease, protein-aggregation-relatedneurodegeneration, Machado-Joseph's disease, spongiform encephalopathy,prion-related disease, multiple sclerosis (MS), progressive supranuclearpalsy (Steel-Richardson-Olszewski disease), Hallervorden-Spatz disease,progressive familial myoclonic epilepsy, cerebellar degeneration, motorneuron disease, Werdnig-Hoffman disease, Wohlfart-Kugelberg-Welanderdisease, Charcot-Marie-Tooth disease, Dejerine-Sottas disease, retinitispigmentosa, Leber's disease, progressive systemic sclerosis,dermatomyositis, and mixed connective tissue disease.

In some embodiments, the neurodegenerative pathology is an acute orchronic degenerative disease of the eye. Acute or chronic degenerativediseases of the eye include glaucoma, dry age-related maculardegeneration, retinitis pigmentosa and other forms of heredodegenerativeretinal disease, retinal detachment, macular pucker, ischemia affectingthe outer retina, cellular damage associated with diabetic retinopathyand retinal ischemia, damage associated with laser therapy, ocularneovascular, diabetic retinopathy, rubeosis iritis, uveitis, Fuch'sheterochromatic iridocyclitis, neovascular glaucoma, cornealneovascularization, retinal ischemia, choroidal vascular insufficinency,choroidal thrombosis, carotid artery ischemia, contusive ocular injury,retinopathy of permaturity, retinal vein occlusion, proliferativevitreoretinopathy, corneal angiogenesis, retinal microvasculopathy, andretinal edema.

In some embodiments, the condition or disorder mediated by HDACcomprises a fibrotic disease such as liver fibrosis, cystic fibrosis,cirrhosis, and fibrotic skin diseases, e.g., hypertrophic scars, keloid,and Dupuytren's contracture. Accordingly, also provided is a method oftreating a fibrotic disease mediated by HDAC in a subject in need ofsuch a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a psychological disorder, such as depression, bipolar diseaseand dementia. In some embodiments, the condition or disorder mediated byHDAC comprises depression. Accordingly, also provided is a method oftreating a psychological disorder, such as depression, mediated by HDACin a subject in need of such a treatment, comprising administering tothe subject a therapeutically effective amount of at least one compound,or pharmaceutically acceptable salt thereof, described herein. In someembodiments, the depression is chosen from major depressive disorder,and bipolar disorder.

In some embodiments, the condition or disorder mediated by HDACcomprises anxiety. Accordingly, also provided is a method of treating ananxiety mediated by HDAC in a subject in need of such a treatment,comprising administering to the subject a therapeutically effectiveamount of at least one compound, or pharmaceutically acceptable saltthereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises schizophrenia. Accordingly, also provided is a method oftreating a schizophrenia mediated by HDAC in a subject in need of such atreatment, comprising administering to the subject a therapeuticallyeffective amount of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a motor neuron disease, muscle atrophy/muscle wastingdisorders, or amyotrophic lateral sclerosis (ALS). Accordingly, alsoprovided is a method of treating a motor neuron disease, muscleatrophy/muscle wasting disorders, or amyotrophic lateral sclerosis (ALS)mediated by HDAC in a subject in need of such a treatment, comprisingadministering to the subject a therapeutically effective amount of atleast one compound, or pharmaceutically acceptable salt thereof,described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a cardiovascular condition. Accordingly, also provided is amethod of treating a cardiovascular condition mediated by HDAC in asubject in need of such a treatment, comprising administering to thesubject a therapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein. In someembodiments, the cardiovascular condition is chosen from cardiomyopathy,cardiac hypertrophy, myocardial ischemia, heart failure, cardiacrestenosis, and arteriosclerosis.

In some embodiments, the condition or disorder mediated by HDACcomprises cancer. Accordingly, also provided is a method of treatingcancer mediated by HDAC in a subject in need of such a treatment,comprising administering to the subject a therapeutically effectiveamount of at least one compound, or pharmaceutically acceptable saltthereof, described herein. In some embodiments, the cancer is chosenfrom lymphoma, pancreatic cancer, colorectal cancer, hepatocellularcarcinoma, Waldenstrom macroglobulinemia, hormone refractory cancer ofthe prostate, and leukaemia, breast cancer, lung cancer, ovarian cancer,prostate cancer, head and neck cancer, renal cancer, gastric cancer,brain cancer, B-cell lymphoma, peripheral T-cell lymphoma, and cutaneousT-cell lymphoma. In some further embodiments, the cancer is chosen fromthe following cancer types. Cardiac: sarcoma (angiosarcoma,fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma,fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamouscell, undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma,hepatocellular adenoma, hemangioma; Bone: osteogenic sarcoma(osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma,chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cellsarcoma), multiple myeloma, malignant giant cell tumor chordoma,osteochronfroma (osteocartilaginous exostoses), benign chondroma,chondroblastoma, chondromyxofibroma, osteoid osteoma and giant celltumors; Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma,osteitis deformans), meninges (meningioma, meningiosarcoma,gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma,germinoma [pinealoma], glioblastoma multiform, oligodendroglioma,schwannoma, retinoblastoma, congenital tumors), spinal cordneurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus(endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervicaldysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma,mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecalcell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignantteratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma,adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma,squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma),fallopian tubes (carcinoma); Hematologic: blood (myeloid leukemia [acuteand chronic], acute lymphoblastic leukemia, chronic lymphocyticleukemia, myeloproliferative diseases, multiple myeloma, myelodysplasticsyndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignantlymphoma]; Skin: malignant melanoma, basal cell carcinoma, squamous cellcarcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma,dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma;and the sensitization of tumors to radiotherapy by administering thecompound according to the invention before, during or after irradiationof the tumor for treating cancer.

In some embodiments, the condition or disorder mediated by HDACcomprises a condition or disorder treatable by immune modulation.Accordingly, also provided is a method of treating a condition ordisorder treatable by immune modulation mediated by HDAC in a subject inneed of such a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein. In someembodiments, the condition or disorder treatable by immune modulation ischosen from asthma, irritable bowel syndrome, Crohn's disease,ulcerative colitis, bowel motility disorders, hypertension, rheumatoidarthritis, osteoarthritis, juvenile chronic arthritis, graft versus hostdisease, psoriasis, spondyloarthropathy, inflammatory bowel disease,alcoholic hepatitis, Sjogren's syndrome, ankylosing spondylitis,membranous glomerulopathy, discogenic pain, systemic lupuserythematosus, allergic bowel disease, coeliac disease, bronchitis,cystic fibrosis, rheumatoid spondylitis, osteoarthritis, uveitis,iritis, and conjunctivitis, ischemic bowel disease, psoriasis, eczema,dermatitis, septic arthritis, gout, pseudogout, juvenile arthritis,Still's disease, Henoch-Schonlein purpura, psoriatic arthritis, myalgia,reactive arthritis (Reiter's syndrome), hemochromatosis, Wegener'sgranulomatosis, familial Mediterranean fever (FMF), HBDS(hyperimmunoglobulinemia D and periodic fever syndrome), TRAPS(TNF-alpha receptor associated periodic fever syndrome), chronicobstructive pulmonary disease, neonatal-onset multisystem inflammatorydisease (NOMID), cryopyrin-associated periodic syndrome (CAPS), andfamilial cold autoinflammatory syndrome (FCAS).

In some embodiments, the condition or disorder mediated by HDACcomprises an allergic disease. Accordingly, also provided is a method oftreating an allergic disease, mediated by HDAC in a subject in need ofsuch a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein. Allergicdiseases include, but are not limited to, respiratory allergic diseasessuch as allergic rhinitis, hypersensitivity lung diseases,hypersensitivity pneumonitis, eosinophilic pneumonias, Loeffler'ssyndrome, chronic eosinophilic pneumonia, delayed-type hypersensitivity,interstitial lung diseases (ILD), idiopathic pulmonary fibrosis,polymyositis, dermatomyositis, systemic anaphylaxis, drug allergies(e.g., to penicillin or cephalosporins), and insect sting allergies.

In some embodiments, the condition or disorder mediated by HDACcomprises an infectious disease such as a fungal infection, bacterialinfection, viral infection, and protozoal infection, e.g., malaria,giardiasis, leishmaniasis, Chaga's disease, dysentery, toxoplasmosis,and coccidiosis. In some embodiments, the condition or disorder mediatedby HDAC comprises malaria. Accordingly, also provided is a method oftreating an infectious disease, such as malaria, mediated by HDAC in asubject in need of such a treatment, comprising administering to thesubject a therapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises autism or Rett syndrome. Accordingly, also provided is amethod of treating autism or Rett syndrome mediated by HDAC in a subjectin need of such a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a hematological disorder such as thalassemia, anemia, andsickle cell anemia. Accordingly, also provided is a method of treating ahematological disorder mediated by HDAC in a subject in need of such atreatment, comprising administering to the subject a therapeuticallyeffective amount of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a metabolic disease such as prediabetes or diabetes (type I orII). Accordingly, also provided is a method of treating a metabolicdisease, such as prediabetes or diabetes (type I or II), mediated byHDAC in a subject in need of such a treatment, comprising administeringto the subject a therapeutically effective amount of at least onecompound, or pharmaceutically acceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a disorder that may also be treated by progenitor/stem cellbased therapies such as: disorders related to diabetes (organ failure,cirrhosis, and hepatitis); central nervous system (CNS) disordersassociated with dysregulation of progenitor cells in the brain (e.g.,post-traumatic stress disorder (PTSD); tumors (e.g., retinoblastomas);disorders affecting oligodendrocyte progenitor cells (e.g., astrocytomasand ependimal cell tumors); multiple sclerosis; demyelinating disorderssuch as the leukodystrophies; neuropathies associated with white matterloss; and cerebellar disorders such as ataxia; and olfactory progenitordisorders (e.g., anosmic conditions). Accordingly, also provided is amethod of treating a disorder that is mediated by HDAC in a subject inneed of such a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein, eitherbefore, during, or after a treatment with progenitor/stem cell basedtherapies.

In some embodiments, the condition or disorder mediated by HDACcomprises a disorder related to the proliferation of epithelial andmesenchymal cells (e.g., tumors, wound healing, and surgeries).Accordingly, also provided is a method of treating a disorder related tothe proliferation of epithelial and mesenchymal cells that is mediatedby HDAC in a subject in need of such a treatment, comprisingadministering to the subject a therapeutically effective amount of atleast one compound, or pharmaceutically acceptable salt thereof,described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a disorder related to the proliferation of bone progenitors(e.g., osteoblasts and osteoclasts), disorders related to hair andepidermal progenitors (e.g., hair loss, cutaneous tumors, skinregeneration, burns, and cosmetic surgery); and disorders related tobone loss during menopause. Accordingly, also provided is a method oftreating disorders related to the proliferation of bone progenitors,disorders related to hair and epidermal progenitors, or disordersrelated to bone loss that are mediated by HDAC in a subject in need ofsuch a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDAC is aviral disorder for which blood cells become sensitized to othertreatments after HDAC inhibition, following administering to the subjecta therapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, as described herein.Accordingly, also provided is a method of treating a viral disorder,wherein blood cells become sensitized to other treatments after HDACinhibition, that is mediated by HDAC in a subject in need of such atreatment, comprising administering to the subject a therapeuticallyeffective amount of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDAC is animmune disorder that may be co-treated with TNFα or other immunemodulators, upon administering to the subject a therapeuticallyeffective amount of at least one compound, or pharmaceuticallyacceptable salt thereof, as described herein. Accordingly, also providedis a method of treating an immune disorder that is mediated by HDAC in asubject in need of such a treatment, comprising administering to thesubject a therapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein, eitherbefore, during, or after a treatment with TNFα or other immunemodulators.

In some embodiments, the condition or disorder mediated by HDACcomprises a graft rejection or transplant rejection. Accordingly, alsoprovided is a method of treating a disorder related to a graft rejectionor a transplant rejection that is mediated by HDAC in a subject in needof such a treatment, comprising administering to the subject atherapeutically effective amount of at least one compound, orpharmaceutically acceptable salt thereof, described herein.

In some embodiments, the condition or disorder mediated by HDACcomprises a blood pressure disorder related to nitric oxide (NO)regulation (e.g., hypertension, erectile dysfunction, asthma; and oculardisorders as glaucoma). Accordingly, also provided is a method oftreating a blood pressure disorder related to nitric oxide (NO)regulation that is mediated by HDAC in a subject in need of such atreatment, comprising administering to the subject a therapeuticallyeffective amount of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein. In some embodiments, thecondition or disorder is a cardiac hypertrophic disorder. Accordingly,also provided is a method of treating a cardiac hypertrophic disorderthat is mediated by HDAC in a subject in need of such a treatment,comprising administering to the subject a therapeutically effectiveamount of at least one compound, or pharmaceutically acceptable saltthereof, described herein.

Also provided are methods of treatment in which at least one compound,or pharmaceutically acceptable salt thereof, described herein is theonly active agent given to the subject and methods of treatment in whichat least one compound, or pharmaceutically acceptable salt thereof,described herein is given to the subject in combination with one or moreadditional active agents.

In general, the compounds, or pharmaceutically acceptable salts thereof,described herein will be administered in a therapeutically effectiveamount by any of the accepted modes of administration for agents thatserve similar utilities. The actual amount of the compound, i.e., theactive ingredient, will depend upon numerous factors such as theseverity of the disease to be treated, the age and relative health ofthe subject, the potency of the compound used, the route and form ofadministration, and other factors well know to the skilled artisan. Thedrug can be administered at least once a day, such as once or twice aday.

In some embodiments, the compounds, or pharmaceutically acceptable saltsthereof, described herein are administered as a pharmaceuticalcomposition. Accordingly, provided are pharmaceutical compositionscomprising at least one compound, or pharmaceutically acceptable saltthereof, described herein, together with at least one pharmaceuticallyacceptable vehicle chosen from carriers, adjuvants, and excipients. Acompound of the present invention can be formulated into pharmaceuticalcompositions using techniques well known to those in the art.

Pharmaceutically acceptable vehicles must be of sufficiently high purityand sufficiently low toxicity to render them suitable for administrationto the animal being treated. The vehicle can be inert or it can possesspharmaceutical benefits. The amount of vehicle employed in conjunctionwith the compound, or pharmaceutically acceptable salt thereof, issufficient to provide a practical quantity of material foradministration per unit dose of the compound, or pharmaceuticallyacceptable salt thereof.

Exemplary pharmaceutically acceptable carriers or components thereof aresugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose, and methyl cellulose; powderedtragacanth; malt; gelatin; talc; solid lubricants, such as stearic acidand magnesium stearate; calcium sulfate; synthetic oils; vegetable oils,such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil;polyols such as propylene glycol, glycerine, sorbitol, mannitol, andpolyethylene glycol; alginic acid; phosphate buffer solutions;emulsifiers, such as the TWEENs®; wetting agents, such sodium laurylsulfate; coloring agents; flavoring agents; tableting agents;stabilizers; antioxidants; preservatives; pyrogen-free water; isotonicsaline; and phosphate buffer solutions.

Optional active agents may be included in a pharmaceutical composition,which do not substantially interfere with the activity of the compound,or pharmaceutically acceptable salt thereof, described herein.

Effective concentrations of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein are mixed with a suitablepharmaceutically acceptable vehicle. In instances in which the compound,or pharmaceutically acceptable salt thereof, exhibits insufficientsolubility, methods for solubilizing compounds may be used. Such methodsare known to those of skill in this art, and include, but are notlimited to, using cosolvents, such as dimethylsulfoxide (DMSO), usingsurfactants, such as TWEEN®, or dissolution in aqueous sodiumbicarbonate.

Upon mixing or addition of a compound, or pharmaceutically acceptablesalt thereof, described herein, the resulting mixture may be a solution,suspension, emulsion or the like. The form of the resulting mixturedepends upon a number of factors, including the intended mode ofadministration and the solubility of the compound, or pharmaceuticallyacceptable salt thereof, in the chosen vehicle. The effectiveconcentration sufficient for ameliorating the symptoms of the diseasetreated may be empirically determined.

The compounds, or pharmaceutically acceptable salts thereof, describedherein may be administered orally, topically, parenterally,intravenously, by intramuscular injection, by inhalation or spray,sublingually, transdermally, via buccal administration, rectally, as anophthalmic solution, or by other means, in dosage unit formulations.

Pharmaceutical compositions may be formulated for oral use, such as forexample, tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsions, hard or soft capsules, orsyrups or elixirs. Pharmaceutical compositions intended for oral use maybe prepared according to any method known to the art for the manufactureof pharmaceutical compositions and such compositions may contain one ormore agents, such as sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide pharmaceuticallyelegant and palatable preparations. In some embodiments, oralpharmaceutical compositions contain from 0.1 to 99% of at least onecompound, or pharmaceutically acceptable salt thereof, described herein.In some embodiments, oral pharmaceutical compositions contain at least5% (weight %) of at least one compound, or pharmaceutically acceptablesalt thereof, described herein. Some embodiments contain from 25% to 50%or from 5% to 75% of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein.

Orally administered pharmaceutical compositions also include liquidsolutions, emulsions, suspensions, powders, granules, elixirs,tinctures, syrups, and the like. The pharmaceutically acceptablecarriers suitable for preparation of such compositions are well known inthe art. Oral pharmaceutical compositions may contain preservatives,flavoring agents, sweetening agents, such as sucrose or saccharin,taste-masking agents, and coloring agents.

Typical components of carriers for syrups, elixirs, emulsions andsuspensions include ethanol, glycerol, propylene glycol, polyethyleneglycol, liquid sucrose, sorbitol and water. Syrups and elixirs may beformulated with sweetening agents, for example glycerol, propyleneglycol, sorbitol or sucrose. Such pharmaceutical compositions may alsocontain a demulcent.

The compound, or pharmaceutically acceptable salt thereof, describedherein can be incorporated into oral liquid preparations such as aqueousor oily suspensions, solutions, emulsions, syrups, or elixirs, forexample. Furthermore, pharmaceutical compositions containing thecompound, or pharmaceutically acceptable salt thereof, described hereincan be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations can containconventional additives, such as suspending agents (e.g., sorbitol syrup,methyl cellulose, glucose/sugar, syrup, gelatin, hydroxyethyl cellulose,carboxymethyl cellulose, aluminum stearate gel, and hydrogenated ediblefats), emulsifying agents (e.g., lecithin, sorbitan monooleate, oracacia), non-aqueous vehicles, which can include edible oils (e.g.,almond oil, fractionated coconut oil, silyl esters, propylene glycol andethyl alcohol), and preservatives (e.g., methyl or propylp-hydroxybenzoate and sorbic acid).

For a suspension, typical suspending agents include methylcellulose,sodium carboxymethyl cellulose, AVICEL® RC-591, tragacanth and sodiumalginate; typical wetting agents include lecithin and polysorbate 80;and typical preservatives include methyl paraben and sodium benzoate.

Aqueous suspensions contain the active material(s) in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents; may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol substitute, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan substitute.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example peanut oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide palatable oralpreparations. These pharmaceutical compositions may be preserved by theaddition of an anti-oxidant such as ascorbic acid.

Pharmaceutical compositions may also be in the form of oil-in-wateremulsions. The oily phase may be a vegetable oil, for example olive oilor peanut oil, or a mineral oil, for example liquid paraffin or mixturesof these. Suitable emulsifying agents may be naturally-occurring gums,for example gum acacia or gum tragacanth, naturally-occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan monooleate, and condensation products of the said partialesters with ethylene oxide, for example polyoxyethylene sorbitanmonooleate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

Tablets typically comprise conventional pharmaceutically acceptableadjuvants as inert diluents, such as calcium carbonate, sodiumcarbonate, mannitol, lactose and cellulose; binders such as starch,gelatin and sucrose; disintegrants such as starch, alginic acid andcroscarmelose; lubricants such as magnesium stearate, stearic acid andtalc. Glidants such as silicon dioxide can be used to improve flowcharacteristics of the powder mixture. Coloring agents, such as the FD&Cdyes, can be added for appearance. Sweeteners and flavoring agents, suchas aspartame, saccharin, menthol, peppermint, and fruit flavors, can beuseful adjuvants for chewable tablets. Capsules (including time releaseand sustained release formulations) typically comprise one or more soliddiluents disclosed above. The selection of carrier components oftendepends on secondary considerations like taste, cost, and shelfstability.

Such pharmaceutical compositions may also be coated by conventionalmethods, typically with pH or time-dependent coatings, such that thecompound, or pharmaceutically acceptable salt thereof, is released inthe gastrointestinal tract in the vicinity of the desired topicalapplication, or at various times to extend the desired action. Suchdosage forms typically include, but are not limited to, one or more ofcellulose acetate phthalate, polyvinylacetate phthalate, hydroxypropylmethylcellulose phthalate, ethyl cellulose, Eudragit® coatings, waxesand shellac.

Pharmaceutical compositions for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example, calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example peanut oil, liquidparaffin or olive oil.

Pharmaceutical compositions may be in the form of a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents that have been mentioned above. The sterileinjectable preparation may also be sterile injectable solution orsuspension in a non-toxic parentally acceptable vehicle, for example asa solution in 1,3-butanediol. Among the acceptable vehicles that may beemployed are water, Ringer's solution, and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid can be useful in the preparation ofinjectables.

The compound, or pharmaceutically acceptable salt thereof, describedherein may be administered parenterally in a sterile medium. Parenteraladministration includes subcutaneous injections, intravenous,intramuscular, intrathecal injection or infusion techniques. Thecompound, or pharmaceutically acceptable salt thereof, described herein,depending on the vehicle and concentration used, can either be suspendedor dissolved in the vehicle. Advantageously, adjuvants such as localanesthetics, preservatives and buffering agents can be dissolved in thevehicle. In many pharmaceutical compositions for parenteraladministration the carrier comprises at least 90% by weight of the totalcomposition. In some embodiments, the carrier for parenteraladministration is chosen from propylene glycol, ethyl oleate,pyrrolidone, ethanol, and sesame oil.

The compound, or pharmaceutically acceptable salt thereof, describedherein may also be administered in the form of suppositories for rectaladministration of the drug. These pharmaceutical compositions can beprepared by mixing the drug with a suitable non-irritating excipientthat is solid at ordinary temperatures but liquid at rectal temperatureand will therefore melt in the rectum to release the drug. Suchmaterials include cocoa butter and polyethylene glycols.

The compound, or pharmaceutically acceptable salt thereof, describedherein may be formulated for local or topical application, such as fortopical application to the skin and mucous membranes, such as in theeye, in the form of gels, creams, and lotions and for application to theeye. Topical pharmaceutical compositions may be in any form including,for example, solutions, creams, ointments, gels, lotions, milks,cleansers, moisturizers, sprays, skin patches, and the like.

Such solutions may be formulated as 0.01%-10% isotonic solutions, pH5-7, with appropriate salts. The compound, or pharmaceuticallyacceptable salt thereof, described herein may also be formulated fortransdermal administration as a transdermal patch.

Topical pharmaceutical compositions comprising at least one compound, orpharmaceutically acceptable salt thereof, described herein can beadmixed with a variety of carrier materials well known in the art, suchas, for example, water, alcohols, aloe vera gel, allantoin, glycerine,vitamin A and E oils, mineral oil, propylene glycol, PPG-2 myristylpropionate, and the like.

Other materials suitable for use in topical carriers include, forexample, emollients, solvents, humectants, thickeners and powders.Examples of each of these types of materials, which can be used singlyor as mixtures of one or more materials, are as follows.

Representative emollients include stearyl alcohol, glycerylmonoricinoleate, glyceryl monostearate, propane-1,2-diol,butane-1,3-diol, mink oil, cetyl alcohol, iso-propyl isostearate,stearic acid, iso-butyl palmitate, isocetyl stearate, oleyl alcohol,isopropyl laurate, hexyl laurate, decyl oleate, octadecan-2-ol, isocetylalcohol, cetyl palmitate, dimethylpolysiloxane, di-n-butyl sebacate,iso-propyl myristate, iso-propyl palmitate, iso-propyl stearate, butylstearate, polyethylene glycol, triethylene glycol, lanolin, sesame oil,coconut oil, arachis oil, castor oil, acetylated lanolin alcohols,petroleum, mineral oil, butyl myristate, isostearic acid, palmitic acid,isopropyl linoleate, lauryl lactate, myristyl lactate, decyl oleate, andmyristyl myristate; propellants, such as propane, butane, iso-butane,dimethyl ether, carbon dioxide, and nitrous oxide; solvents, such asethyl alcohol, methylene chloride, iso-propanol, castor oil, ethyleneglycol monoethyl ether, diethylene glycol monobutyl ether, diethyleneglycol monoethyl ether, dimethyl sulphoxide, dimethyl formamide,tetrahydrofuran; humectants, such as glycerin, sorbitol, sodium2-pyrrolidone-5-carboxylate, soluble collagen, dibutyl phthalate, andgelatin; and powders, such as chalk, talc, fullers earth, kaolin,starch, gums, colloidal silicon dioxide, sodium polyacrylate, tetraalkyl ammonium smectites, trialkyl aryl ammonium smectites, chemicallymodified magnesium aluminium silicate, organically modifiedmontmorillonite clay, hydrated aluminium silicate, fumed silica,carboxyvinyl polymer, sodium carboxymethyl cellulose, and ethyleneglycol monostearate.

The compound, or pharmaceutically acceptable salt thereof, describedherein may also be topically administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles, and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Other pharmaceutical compositions useful for attaining systemic deliveryof the compound, or pharmaceutically acceptable salt thereof, includesublingual, buccal and nasal dosage forms. Such pharmaceuticalcompositions typically comprise one or more of soluble filler substancessuch as sucrose, sorbitol and mannitol, and binders such as acacia,microcrystalline cellulose, carboxymethyl cellulose, and hydroxypropylmethylcellulose. Glidants, lubricants, sweeteners, colorants,antioxidants and flavoring agents disclosed above may also be included.

Pharmaceutical compositions for inhalation typically can be provided inthe form of a solution, suspension or emulsion that can be administeredas a dry powder or in the form of an aerosol using a conventionalpropellant (e.g., dichlorodifluoromethane or trichlorofluoromethane).

The pharmaceutical compositions may also optionally comprise an activityenhancer. The activity enhancer can be chosen from a wide variety ofmolecules that function in different ways to enhance or be independentof therapeutic effects of the compound, or pharmaceutically acceptablesalt thereof, described herein. Particular classes of activity enhancersinclude skin penetration enhancers and absorption enhancers.

Pharmaceutical compositions may also contain additional active agentsthat can be chosen from a wide variety of molecules, which can functionin different ways to enhance the therapeutic effects of at least onecompound, or pharmaceutically acceptable salt thereof, described herein.These optional other active agents, when present, are typically employedin the pharmaceutical compositions at a level ranging from 0.01% to 15%.Some embodiments contain from 0.1% to 10% by weight of the composition.Other embodiments contain from 0.5% to 5% by weight of the composition.

Also provided are packaged pharmaceutical compositions. Such packagedcompositions include a pharmaceutical composition comprising at leastone compound, or pharmaceutically acceptable salt thereof, describedherein, and instructions for using the composition to treat a subject(typically a human patient). In some embodiments, the instructions arefor using the pharmaceutical composition to treat a subject suffering acondition or disorder mediated by HDAC. The packaged pharmaceuticalcomposition can include providing prescribing information; for example,to a patient or health care provider, or as a label in a packagedpharmaceutical composition. Prescribing information may include forexample efficacy, dosage and administration, contraindication andadverse reaction information pertaining to the pharmaceuticalcomposition.

In all of the foregoing the compound, or pharmaceutically acceptablesalt thereof, can be administered alone, as mixtures, or in combinationwith other active agents.

The methods described herein include methods for treating Huntington'sdisease, including treating memory and/or cognitive impairmentassociated with Huntington's disease, comprising administering to asubject, simultaneously or sequentially, at least one compound, orpharmaceutically acceptable salt thereof, described herein and one ormore additional agents used in the treatment of Huntington's diseasesuch as, but not limited to, Amitriptyline, Imipramine, Desipramine,Nortriptyline, Paroxetine, Fluoxetine, Sertraline, Tetrabenazine,Haloperidol, Chlorpromazine, Thioridazine, Sulpride, Quetiapine,Clozapine, and Risperidone. In methods using simultaneousadministration, the agents can be present in a combined composition orcan be administered separately. As a result, also provided arepharmaceutical compositions comprising at least one compound, orpharmaceutically acceptable salt thereof, described herein and one ormore additional pharmaceutical agents used in the treatment ofHuntington's disease such as, but not limited to, Amitriptyline,Imipramine, Desipramine, Nortriptyline, Paroxetine, Fluoxetine,Sertraline, Tetrabenazine, Haloperidol, Chlorpromazine, Thioridazine,Sulpride, Quetiapine, Clozapine, and Risperidone. Similarly, alsoprovided are packaged pharmaceutical compositions containing apharmaceutical composition comprising at least one compound, orpharmaceutically acceptable salt thereof, described herein, and anothercomposition comprising one or more additional pharmaceutical agents usedin the treatment of Huntington's disease such as, but not limited to,Amitriptyline, Imipramine, Desipramine, Nortriptyline, Paroxetine,Fluoxetine, Sertraline, Tetrabenazine, Haloperidol, Chlorpromazine,Thioridazine, Sulpride, Quetiapine, Clozapine, and Risperidone.

Also provided are methods for treating Alzheimer's disease, includingtreating memory and/or cognitive impairment associated with Alzheimer'sdisease, comprising administering to a subject, simultaneously orsequentially, at least one compound, or pharmaceutically acceptable saltthereof, described herein and one or more additional agents used in thetreatment of Alzheimer's disease such as, but not limited to, Reminyl®,Cognex®, Aricept®, Exelon®, Akatinol®, Neotropin™, Eldepryl®, Estrogenand Clioquinol. In methods using simultaneous administration, the agentscan be present in a combined composition or can be administeredseparately. Also provided are pharmaceutical compositions comprising atleast one compound, or pharmaceutically acceptable salt thereof,described herein, and one or more additional pharmaceutical agents usedin the treatment of Alzheimer's disease such as, but not limited to,Reminyl®, Cognex®, Aricept®, Exelon®, Akatinol®, Neotropin™, Eldepryl®,Estrogen and Clioquinol. Similarly, also provided are packagedpharmaceutical compositions containing a pharmaceutical compositioncomprising at least one compound, or pharmaceutically acceptable saltthereof, described herein, and another composition comprising one ormore additional pharmaceutical agents used in the treatment ofAlzheimer's disease such as, but not limited to Reminyl®, Cognex®,Aricept®, Exelon®, Akatinol®, Neotropin™, Eldepryl®, Estrogen andClioquinol.

Also provided are methods for treating cancer comprising administeringto a subject, simultaneously or sequentially, at least one compound, orpharmaceutically acceptable salt thereof, described herein and one ormore additional agents used in the treatment of cancer such as, but notlimited to, the following categories of anti-tumor agents:

(i) other cell cycle inhibitory agents that work by the same ordifferent mechanisms from those defined hereinbefore, for example cyclindependent kinase (CDK) inhibitors, in particular CDK2 inhibitors;

(ii) cytostatic agents such as antioestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene, iodoxyfene), progestogens (forexample megestrol acetate), aromatase inhibitors (for exampleanastrozole, letrazole, vorazole, exemestane), antiprogestogens,antiandrogens (for example flutamide, nilutamide, bicalutamide,cyproterone acetate), LHRH agonists and antagonists (for examplegoserelin acetate, luprolide), inhibitors of testosterone5α-dihydroreductase (for example finasteride), anti-invasion agents (forexample metalloproteinase inhibitors like marimastat and inhibitors ofurokinase plasminogen activator receptor function) and inhibitors ofgrowth factor function, (such growth factors include for examplevascular endothelial growth factor, epithelial growth factor, plateletderived growth factor and hepatocyte growth factor such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies,tyrosine kinase inhibitors and serine/threonine kinase inhibitors);

(iii) antiproliferative/antineoplastic drugs and combinations thereof,as used in medical oncology, such as antimetabolites (for exampleantifolates like methotrexate, fluoropyrimidines like 5-fluorouracil,purine and adenosine analogues, cytosine arabinoside); antitumourantibiotics (for example anthracyclines like doxorubicin, daunomycin,epirubicin and idarubicin, mitomycin-C, dactinomycin, mithramycin);platinum derivatives (for example cisplatin, carboplatin); alkylatingagents (for example nitrogen mustard, melphalan, chlorambucil,busulphan, cyclophosphamide, ifosfamide, nitrosoureas, thiotepa);antimitotic agents (for example vinca alkaloids like vincrisitine andtaxoids like taxol, taxotere); topoisomerase inhibitors (for exampleepipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan);

(iv) antiangiogenic agents that work by different mechanisms from thosedefined hereinbefore (for example receptor tyrosine kinases like Tie-2,inhibitors of integrin αvβ₃ function, angiostatin, razoxin,thalidomide), and including vascular targeting agents; and

(v) differentiation agents (for example retinoic acid and vitamin D).

In methods using simultaneous administration, the agents can be presentin a combined composition or can be administered separately. Alsoprovided are pharmaceutical compositions comprising at least onecompound, or pharmaceutically acceptable salt thereof, described herein,and one or more anti-tumor agent as described herein. Similarly, alsoprovided are packaged pharmaceutical compositions containing apharmaceutical composition comprising at least one compound, orpharmaceutically acceptable salt thereof, described herein, and anothercomposition comprising one or more one or more anti-tumor agent asdescribed herein. When used in combination with one or more additionalpharmaceutical agent or agents, the described herein may be administeredprior to, concurrently with, or following administration of theadditional pharmaceutical agent or agents.

In some embodiments, the compounds, or pharmaceutically acceptable saltsthereof, described herein, are administered in conjunction with surgeryor radiotherapy, optionally in combination with one or more additionalagents used in the treatment of cancer.

The dosages of the compounds described herein depend upon a variety offactors including the particular syndrome to be treated, the severity ofthe symptoms, the route of administration, the frequency of the dosageinterval, the particular compound utilized, the efficacy, toxicologyprofile, pharmacokinetic profile of the compound, and the presence ofany deleterious side-effects, among other considerations.

The compound, or pharmaceutically acceptable salt thereof, describedherein is typically administered at dosage levels and in a mannercustomary for HDAC inhibitors. For example, the compound, orpharmaceutically acceptable salt thereof, can be administered, in singleor multiple doses, by oral administration at a dosage level of generally0.001-100 mg/kg/day, for example, 0.01-100 mg/kg/day, such as 0.1-70mg/kg/day, for example, 0.5-10 mg/kg/day. Unit dosage forms can containgenerally 0.01-1000 mg of at least one compound, or pharmaceuticallyacceptable salt thereof, described herein, for example, 0.1-50 mg of atleast one compound, or pharmaceutically acceptable salt thereof,described herein. For intravenous administration, the compounds can beadministered, in single or multiple dosages, at a dosage level of, forexample, 0.001-50 mg/kg/day, such as 0.001-10 mg/kg/day, for example,0.01-1 mg/kg/day. Unit dosage forms can contain, for example, 0.1-10 mgof at least one compound, or pharmaceutically acceptable salt thereof,described herein.

A labeled form of a compound, or pharmaceutically acceptable saltthereof, described herein can be used as a diagnostic for identifyingand/or obtaining compounds that have the function of modulating anactivity of HDAC as described herein. The compound, or pharmaceuticallyacceptable salt thereof, described herein may additionally be used forvalidating, optimizing, and standardizing bioassays.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescent tag, enzyme, antibodies, particles suchas magnetic particles, chemiluminescent tag, or specific bindingmolecules, etc. Specific binding molecules include pairs, such as biotinand streptavidin, digoxin and antidigoxin etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

The present disclosure includes all isotopes of atoms occurring in thecompounds and pharmaceutically acceptable salts thereof describedherein. Isotopes include those atoms having the same atomic number butdifferent mass numbers. The present disclosure also includes everycombination of one or more atoms in the compounds and pharmaceuticallyacceptable salts thereof described herein that is replaced with an atomhaving the same atomic number but a different mass number. One suchexample is the replacement of an atom that is the most naturallyabundant isotope, such as ¹H or ¹²C, found in one of the compounds andpharmaceutically acceptable salts thereof described herein, with adifferent atom that is not the most naturally abundant isotope, such as²H or ³H (replacing ¹H), or ¹¹C, ¹³C, or ¹²C (replacing ¹²C). A compoundwherein such a replacement has taken place is commonly referred to asbeing isotopically-labeled. Isotopic-labeling of the compounds andpharmaceutically acceptable salts thereof described herein can beaccomplished using any one of a variety of different synthetic methodsknow to those of ordinary skill in the art and they are readily creditedwith understanding the synthetic methods and available reagents neededto conduct such isotopic-labeling. By way of general example, andwithout limitation, isotopes of hydrogen include ²H (deuterium) and ³H(tritium). Isotopes of carbon include ¹¹C, ¹³C, and ¹⁴C. Isotopes ofnitrogen include ¹³N and ¹⁵N. Isotopes of oxygen include ¹⁵O, ¹⁷O, and¹⁸O. An isotope of fluorine includes ¹⁸F. An isotope of sulfur includes³⁵S. An isotope of chlorine includes ³⁶Cl. Isotopes of bromine include⁷⁵Br, ⁷⁶Br, ⁷⁷Br, and ⁸²Br. Isotopes of iodine include ¹²³I, ¹²⁴I, ¹²⁵I,and ¹³¹I. Also provided are pharmaceutical compositions comprising acompound or a pharmaceutically acceptable salt thereof described herein,wherein the naturally occurring distribution of the isotopes in thepharmaceutical composition is perturbed. Also provided arepharmaceutical compositions comprising a compound or a pharmaceuticallyacceptable salt thereof described herein enriched at one or morepositions with an isotope other than the most naturally abundantisotope. Methods are readily available to measure such isotopeperturbations or enrichments, such as, mass spectrometry, and forisotopes that are radio-isotopes additional methods are available, suchas, radio-detectors used in connection with HPLC or GC. Certainisotopically-labeled compounds and pharmaceutically acceptable saltsthereof described herein are useful in compound and/or substrate tissuedistribution assays. In some embodiments the radionuclide ³H and/or ¹⁴Cisotopes are useful in these studies. Further, substitution with heavierisotopes such as deuterium (i.e., ²H) may afford certain therapeuticadvantages resulting from greater metabolic stability (e.g., increasedin vivo half-life or reduced dosage requirements) and hence may bepreferred in some circumstances. Isotopically labeled compounds andpharmaceutically acceptable salts thereof described herein can generallybe prepared by following procedures analogous to those disclosed in theExamples infra, by substituting an isotopically labeled reagent for anon-isotopically labeled reagent. Moreover, it should be understood thatall of the atoms represented in the compounds and pharmaceuticallyacceptable salts thereof described herein can be either the mostcommonly occurring isotope of such atoms or a scarcer radio-isotope ornonradioactive isotope.

In carrying out the procedures of the methods described herein, it is ofcourse to be understood that reference to particular buffers, media,reagents, cells, culture conditions and the like are not intended to belimiting, but are to be read so as to include all related materials thatone of ordinary skill in the art would recognize as being of interest orvalue in the particular context in which that discussion is presented.For example, it is often possible to substitute one buffer system orculture medium for another and still achieve similar, if not identical,results. Those of skill in the art will have sufficient knowledge ofsuch systems and methodologies so as to be able, without undueexperimentation, to make such substitutions as will optimally servetheir purposes in using the methods and procedures disclosed herein.

EXAMPLES

The compounds, or pharmaceutically acceptable salts thereof,compositions, and methods described herein are further illustrated bythe following non-limiting examples.

As used herein, the following abbreviations have the following meanings.If an abbreviation is not defined, it has its generally acceptedmeaning.

ABBREVIATIONS

-   aq.: Aqueous-   AUC: Area under curve-   [bmim][PF₆]: 1-Butyl-3-methylimidazolium hexafluorophosphate-   BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium    hexafluorophosphate-   conc.: Concentrated-   d: Doublet-   DCM: Dichloromethane-   DCE: Dichloroethane-   DIPEA: Diisopropylethylamine-   DMA: Dimethylacetamide-   DME: Dimethoxyethane-   DMF: Dimethylformamide-   DMSO: Dimethylsulfoxide-   eq.: Equivalent-   ES+: Electrospray Positive Ionisation-   ES−: Electrospray Negative Ionisation-   Et₂O: Diethyl ether-   EtOAc: Ethyl acetate-   g: Gram-   h: Hour-   HPLC: High Performance Liquid Chromatography-   Hz: Hertz-   IV: Intravenous-   J: Coupling constant-   kg: Kilogram-   LCMS: Liquid Chromatography Mass Spectrometry-   LiHMDS: Lithium bis(trimethylsilyl)amide-   m: Multiplet-   M: Mass-   MeCN: Acetonitrile-   MeOH: Methanol-   mg: Milligram-   min: Minute-   mL: Milliliter-   mmol: Millimole-   ng: Nanogram-   nM: Nanomolar-   NMP: N-Methyl pyrrolidinone-   N.M.R.: Nuclear magnetic resonance-   Pd/C: Palladium on carbon-   Pd₂(dba)₃: Tris(dibenzylideneacetone)dipalladium(0)-   Pd(dppf)Cl₂:    [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)-   Pd(PPh₃)₄: Tetrakis(triphenylphosphine)palladium(O)-   PO: Oral-   o-tol: ortho-Tolyl-   Rf: Retention factor-   Rh₂(OAc)₄: Rhodium(II) acetate-   RT or R_(t): Retention time-   r.t.: Room temperature-   RuPhos: 2-Dicyclohexylphosphino-2′,6′-di-iso-propoxy-1,1′-biphenyl-   s: Singlet-   THF: Tetrahydrofuran-   TMSCN: Trimethylsilyl cyanide-   w/v: Weight to volume-   Xantphos: 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene-   μL: Microliter-   μM: Micromolar

Example 1 Synthesis of methyl2-(4-bromophenyl)-2-(2-fluorophenyl)acetate (4)

a. Synthesis of (4-bromophenyl)(2-fluorophenyl)methanol (1)

This reaction was performed under nitrogen. Magnesium (5.2 g, 214 mmol;1.1 eq.) was covered with ether (40 mL) in a 1 L round bottom flaskequipped with a refluxing condenser and a pressure equalizing droppingfunnel. A solution of 1,4-dibromobenzene (45.8 g, 194 mmol) in ether(200 mL) was added dropwise at a rate sufficient to first initiate andthen sustain reflux over a 1.5 h period. A few crystals of iodine wereused to initiate the reaction. After complete addition, the reactionmixture was stirred at r.t. for 1 h. The mixture was cooled to 0° C.(ice bath) and a solution of 2-fluorobenzaldehyde (22.5 mL, 26.6 g, 214mmol; 1.1 eq.) in ether (45 mL) was added dropwise over 20 min. Themixture was left to warm up to r.t. and was stirred overnight. Thereaction mixture was cooled to 0° C. (ice-bath) and quenched with amixture of aqueous NH₄Cl (50 mL) and 2 M HCl (50 mL). EtOAc (100 mL) wasadded and the mixture was filtered through Celite®, washing with EtOAc(2×50 mL). The two phases were separated and the organic phase waswashed with brine (70 mL), dried (MgSO₄), filtered and concentrated invacuo to give a yellow oil (59 g). Purification by flash columnchromatography using 10% EtOAc in isohexane (400 g of silica; columndiameter: 80 mm) (R_(f) 0.26) gave the subtitle compound as a yellow oil(29.83 g, 55% yield). LCMS(R_(t) 3.17 min, [M-OH]+263/265).

b. Synthesis of 1-((4-bromophenyl)chloromethyl)-2-fluorobenzene (2)

The reaction was carried out under nitrogen. To a solution of compound 1(29.76 g, 105.85 mmol) in DCM (150 mL) cooled to 0° C. (ice-bath) wasadded thionyl chloride (8.5 mL, 13.85 g, 116.44 mmol, 1.1 eq.). Thereaction mixture was left to warm up to r.t. and was stirred for 72 h.The mixture was poured into 2M Na₂CO₃ (150 mL). The two phases wereseparated and the organic layer was washed with brine (2×50 mL), dried(MgSO₄), filtered and concentrated in vacuo to give the subtitlecompound as a yellow oil (29.8 g, 94% yield). LCMS(R_(t) 3.67 min,[M+H]+ 299 not seen). ¹H N.M.R. (CDCl₃) 7.57-7.40 (m, 3H), 7.38-7.28 (m,3H), 7.19-7.15 (m, 1H), 7.07-7.02 (m, 1H), 6.37 (s, 1H).

c. Synthesis of 2-(4-bromophenyl)-2-(2-fluorophenyl)acetonitrile (3)

Compound 2 (29.8 g, 99.62 mmol) was dissolved in dichloromethane (190mL) and the solution was cooled to 0° C. (ice-bath). Trimethylsilylcyanide (12.5 mL, 9.88 g, 99.62 mmol) was added followed by titanium(IV)chloride (10.9 mL, 18.9 g, 99.62 mmol). The mixture was left to warm upto r.t. and was stirred overnight. The mixture was poured into ice-water(˜300 mL) containing Na₂CO₃ (50 g), then diluted with more DCM (˜200 mL)and filtered through Celite. After separation of the two phases theaqueous layer was re-extracted with DCM (200 mL) and the organic phasewas dried (MgSO₄), filtered and concentrated in vacuo to give thesubtitle compound as an orange oil (28.65 g, 99% yield). LCMS(R_(t) 3.38min, [M+H]+ 290/292). ¹H N.M.R. (CDCl₃) 7.52-7.49 (m, 2H), 7.48-7.41 (m,1H), 7.40-7.31 (m, 2H), 7.29-7.25 (m, 1H), 7.25-7.17 (m, 1H), 7.15-7.07(m, 1H), 5.39 (s, 1H).

d. Synthesis of methyl 2-(4-bromophenyl)-2-(2-fluorophenyl)acetate (4)

A solution of compound 3 (23.5 g, 80.99 mmol) and c.H₂SO₄ (24.5 mL) inmethanol (175 mL) was heated under reflux for 20 h. LCMS showed mainly amixture of unreacted starting material (R_(t) 3.38 min, [M+H]+ 290, 51%)and desired product (R_(t) 3.44 min, [M+H]+ 324 not seen, 36%). c.H₂SO₄(8.2 mL) and MeOH (30 mL) were added and the reaction mixture was heatedunder reflux for 72 h. LCMS indicated still some unreacted statingmaterial (27%). More c.H₂SO₄ (8.2 mL) and MeOH (30 mL) were added andthe mixture was heated under reflux for a further 20 h. LCMS showedmainly the desired product (R_(t) 3.44 min, [M+H]+ 324 not seen, 84%).The reaction mixture was cooled to 0° C. (ice-bath) and partitionedbetween H₂O (100 mL) and DCM (400 mL). The two phases were separated andthe organic layer was dried (MgSO₄), filtered and concentrated in vacuoto give a yellow oil (25.81 g). Purification by flash columnchromatography using 3% EtOAc in isohexane (400 g of silica; columndiameter: 80 mm) (R_(f) 0.17) gave the title compound as a pale yellowoil (16.4 g, 63% yield). LCMS(R_(t) 3.44 min, [M+H]+ 324 not seen). ¹HN.M.R. (CDCl₃) 7.48-7.45 (m, 2H), 7.30-7.18 (m, 4H), 7.12-7.03 (m, 2H),5.24 (s, 1H), 3.75 (s, 3H).

Example 2 Synthesis of2-(2-fluorophenyl)-N-hydroxy-2-(4-(2-methylpyrimidin-5-yl)phenyl)acetamide(6)

Synthesis of methyl2-(2-fluorophenyl)-2-(4-(2-methylpyrimidin-5-yl)phenyl)acetate (5)

Yields (after chromatography) of intermediate ester 5, performed atdifferent scales:

0.3 g of aryl bromide (0.9 mmol) gave 5 in 85% yield;

2.0 g of aryl bromide (6.2 mmol) gave 5 in 79% yield;

4.0 g of aryl bromide (12.4 mmol) gave 5 in 79% yield.

To a solution of methyl-2-(4-bromophenyl)-2-(2-fluorophenyl)acetate (4)(4.0 g, 12.4 mmol) in DME/MeOH (80 mL:20 mL) was addedbis(pinacolato)diboron (3.40 g, 13.4 mmol) and CsF (3.80 g, 25.0 mmol).The mixture was degassed with N₂ for 5 min, treated with Pd(PPh₃)₄ (570mg, 0.5 mmol) and heated at 110° C. for 1 h (LCMS indicated completionof the reaction). 5-Bromo-2-methylpyrimidine (2.50 g, 14.4 mmol), CsF(3.80 g, 25.0 mmol) and Pd(PPh₃)₄ (570 mg, 0.5 mmol) were added to themixture and heating was continued at 110° C. for 1 h. The mixture wascooled to r.t., treated with water (100 mL) and extracted with DCM(2×100 mL). The combined organic phase was separated, dried (MgSO₄), andconcentrated in vacuo. Purification by chromatography (SiO₂) elutingwith DCM to remove unreacted boronic esters followed by Et₂O affordedthe subtitle compound as a pale yellow oil (3.3 g, 79% yield). LCMS(R_(t) 2.94 min, [M+H]+ 337) ¹H N.M.R. (CDCl₃) 8.83 (s, 2H), 7.55 (d,2H, J=6.4 Hz), 7.46 (d, 2H, J=6.4 Hz), 7.32-7.29 (m, 2H), 7.15-7.05 (m,2H), 5.34 (s, 1H), 3.79 (s, 3H), 2.79 (s, 3H).

Hydroxamic acid formation:2-(2-fluorophenyl)-N-hydroxy-2-(4-(2-methylpyrimidin-5-yl)phenyl)acetamide

To a stirred solution of ester 5 (5.0 g, 15 mmol) in MeOH (40 mL) wasadded hydroxylamine (10 mL, 50% aqueous solution, 150 mmol) and sodiumhydroxide (1.2 g, 30 mmol). The mixture was stirred at r.t. for 2 h,then treated with saturated ammonium chloride solution and extractedinto ethyl acetate. The combined organic phase was separated, dried(MgSO₄), and evaporated in vacuo. Purification by trituration with 97:3DCM/MeOH gave the title compound as a white solid (3.42 g, 70% yield).

LCMS(R_(t) 2.30 min, [M+H]+ 338). 1H N.M.R. (DMSO-d₆) 11.05 (s, 1H),9.35 (s, 2H), 9.04 (s, 1H), 7.74 (d, 2H, J=8.2 Hz), 7.53 (t, 1H, J=8.2Hz), 7.42 (d, 2H, J=8.2 Hz), 7.38-7.31 (m, 1H), 7.22-7.17 (m, 2H), 5.08(s, 1H), 2.66 (s, 3H).

Example 3 Synthesis of2-(2-fluorophenyl)-N-hydroxy-2-(4-(5-(trifluoromethyl)pyrimidin-2-yl)phenyl)acetamide(8)

Synthesis of methyl2-(2-fluorophenyl)-2-(4-(5-(trifluoromethyl)pyrimidin-2-yl)phenyl)acetate(7)

To a stirred solution ofmethyl-2-(4-bromophenyl)-2-(2-fluorophenyl)acetate (2.0 g, 6.2 mmol) inDME/MeOH (80 mL:20 mL) was added bis(pinacolato)diboron (1.70 g, 7.2mmol) and CsF (1.90 g, 12.5 mmol). The mixture was degassed with N₂ for5 min, treated with Pd(PPh₃)₄ (300 mg, 0.3 mmol) and heated at 110° C.for 1 h. The mixture was cooled to r.t., treated with water (100 mL) andextracted with DCM (2×100 mL). The combined organic phase was separated,dried (MgSO₄), and concentrated in vacuo to yieldmethyl-2-(2-fluorophenyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetateas an orange oil (1.83 g, 80% yield).

To a stirred solution ofmethyl-2-(2-fluorophenyl)-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)acetate(1.83 g, 4.8 mmol) in dioxane (80 mL) and water (20 mL) was added2-chloro-5-trifluoromethylpyrimidine (1.0 g, 5.5 mmol) and K₂CO₃ (1.35g, 9.8 mmol). The mixture was degassed with N₂ for 5 min, treated withPd(PPh₃)₄ (280 mg, 0.24 mmol) and heated at 110° C. overnight (LCMSindicated completion of the reaction). The mixture was cooled to r.t.,treated with water (100 mL) and extracted with DCM (2×100 mL). Thecombined organic phase was separated, dried (MgSO₄), and evaporated invacuo. Purification by chromatography (SiO₂) eluting withisohexane/ethyl acetate (95:5) afforded the subtitle compound as a whitesolid (1.45 g, 74% yield). LCMS(R_(t) 3.54 min, [M+H]+ 391). ¹H N.M.R.(CDCl₃) 9.01 (s, 2H), 8.48 (d, 2H, J=8.4 Hz), 7.47 (d, 2H, J=8.4 Hz),7.30-7.25 (m, 2H), 7.13-7.05 (m, 2H), 5.38 (s, 1H), 3.78 (s, 3H).

Hydroxamic acid formation:2-(2-fluorophenyl)-N-hydroxy-2-(4-(5-(trifluoromethyl)pyrimidin-2-yl)phenyl)acetamide(8)

To a stirred solution ofmethyl-2-(2-fluorophenyl)-2-(4-(5-(trifluoromethyl)pyrimidine-2-yl)phenyl)acetate(6.6 g, 16.9 mmol) in methanol (80 mL) at r.t. was added hydroxylamine(11.0 mL, 50% aqueous solution, 169.0 mmol) and sodium hydroxide (9.0mL, 15% aqueous solution, 33.8 mmol). The mixture was stirred at r.t.for 2.5 h, treated with water (100 mL) and extracted with DCM (2×50 mL).The aqueous layer was concentrated in vacuo to half bulk, neutralizedwith saturated NH₄Cl solution (50 mL) and the resulting precipitate wasfiltered, washed with water and dried. LCMS indicated 80% purity.Recrystallisation from chloroform (40 mL) afforded the title compound asa white solid (5.3 g, 82% yield). LCMS(R_(t)2.94 min, [M+H]+ 392). ¹HN.M.R. (DMSO-d₆) 11.08 (s, 1H), 9.38 (s, 2H), 9.08 (s, 1H), 8.44 (d, 2H,J=8.2 Hz), 7.55-7.49 (m, 3H), 7.37-7.33 (m, 1H), 7.23-7.18 (m, 2H), 5.13(s, 1H).

Example 4 Analysis of Inhibition of HDAC4

The potency of exemplifying compounds descried herein is quantified bymeasuring the Histone Deacetylase 4 (HDAC4) catalytic domain enzymaticactivity using the Class IIa selective substrate, Boc-Lys(Tfa)-AMC. Thesubstrate is deacetylated to Boc-Lys-AMC by HDAC4. Cleavage by trypsinresults in the release of the fluorophore AMC from the deacetylatedsubstrate. The fluorescence of the sample is directly related to thehistone deacetylase activity in the sample.

Serially Dilute Compounds.

Serial dilutions of the compounds to be tested and control referencecompound(1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone)are made by first resuspending the lyophilized compound to a finalconcentration of 10 mM in 100% dimethyl sulfoxide (DMSO). Stocks of 60μL aliquots of the 10 mM compound in DMSO are prepared and stored at−20° C. From one stock aliquot of each compound to be tested and thereference compound, a 16-point serial dilution is prepared according toTable 1 using a 125 μL 16-channel Matrix multi-channel pipette (MatrixTechnologies Ltd).

TABLE 1 Serial Dilution of Compounds Concentration Dilution DilutedSolutions Well (μM) ratio Volumes Concentration 1 A 10000 — 60 μL 10 mMTest compound/reference control Concentration 2 B 5000 1:2 30 μL A + 30μL DMSO Concentration 3 C 2500 1:2 30 μL B + 30 μL DMSO Concentration 4D 1000   1:2.5 30 μL C + 45 μL DMSO Concentration 5 E 500 1:2 30 μL D +30 μL DMSO Concentration 6 F 250 1:2  30 μL E + 30 μL DMSO Concentration7 G 125 1:2  30 μL F + 30 μL DMSO Concentration 8 H 62.5 1:2 30 μL G +30 μL DMSO Concentration 9 I 31.25 1:2 30 μL 1 H + 30 μL DMSO Concentration 10 J 15.63 1:2  30 μL I + 30 μL DMSO Concentration 11 K7.81 1:2  30 μL J + 30 μL DMSO Concentration 12 L 3.91 1:2 30 μL K + 30μL DMSO Concentration 13 M 1.95 1:2  30 μL L + 30 μL DMSO Concentration14 N 0.98 1:2 30 μL M + 30 μL DMSO  Concentration 15 O 0.49 1:2 30 μLN + 30 μL DMSO Concentration 16 P 0.24 1:2 30 μL O + 30 μL DMSO

2 μL (200×) of each diluted solution and each control (full activity:100% DMSO alone or full inhibition 1 mM) is stamped into V-bottomedpolypropylene 384-well compound plates using either the Bravo (384-wellhead from Agilent) or 12.5 μL 16-channel Matrix multi-channel pipette(Matrix Technologies Ltd). Each well with the 200× compound solution isdiluted 1:20 by the addition of 38 μL assay buffer+DMSO (10.5% DMSO, 45mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl, and 0.9 mM MgCl₂ at pH 8.0 andequilibrated to r.t.).

Prepare HDAC4 Catalytic Domain Enzyme (0.286 μg/mL).

The HDAC4 catalytic domain enzyme is human catalytic domain HDAC4protein (amino acids 648-1032 (with a C-terminal 6× histidine tag), madeand provided by BioFocus at 0.5 mg/mL. A working solution of enzyme isprepared from a 0.5 mg/mL stock aliquot of HDAC4 catalytic domain(thawed on ice) diluted to 0.286 μg/mL with assay buffer (50 mMTris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂ at pH 8 andequilibrated to r.t.) just prior to the addition of the enzyme to theassay.

Prepare 5× (50 μM) Boc-Lys(Tfa)-AMC substrate.

5× (50 μM) substrate is prepared just prior to the addition to theassay. A 1 mM substrate stock is made by diluting a 100 mMBoc-Lys(Tfa)-AMC in DMSO solution 1:100 by adding it drop-wise to assaybuffer (equilibrated to r.t.) while vortexing at slow speed to preventprecipitation. The 5× substrate is prepared by diluting the 1 mMsubstrate solution 1:20 by adding it drop-wise to assay buffer(equilibrated to r.t.) while vortexing at slow speed to preventprecipitation.

Prepare 3× (30 μM) Developer/Stop Solution.

3× (30 μM) Developer/Stop Solution is prepared just prior to addition tothe plate by diluting a stock solution of 10 mM reference compound 1:333in 25 mg/mL trypsin (PAA Laboratories Ltd.) equilibrated to r.t.

Assay.

5 μL of each solution of 1:20 diluted compound from above is transferredto a clear bottomed, black, 384-well assay plate using the Bravo or theJanus (384-well MDT head from Perkin Elmer). Using a 16-channel Matrixmulti-channel pipette, 35 μL of the working solution of HDAC4 catalyticdomain enzyme (0.286 μg/mL in assay buffer) is transferred to the assayplate. The assay is then started by adding 10 μL of 5× (50 μM) substrateto the assay plates using either the Bravo, Janus or 16-channel Matrixmulti-channel pipette. The assay plate is then shaken for two minutes onan orbital shaker at 900 rpm (rotations per minute). Next the plate isincubated for 15 minutes at 37° C. The reaction is stopped by adding 25μL of 3× (30 μM) developer/stop solution to the assay plates usingeither the Bravo, Janus or a 16-channel Matrix multi-channel pipette.Assay plates are then shaken for 5 minutes on an orbital shaker at 1200rpm. Next, the assay plates are incubated at 37° C. for 1 hour in atissue culture incubator. Finally, the fluorescence is measured(Excitation: 355 nm, Emission: 460 nm) using PerkinElmer EnVision in topread mode.

Example 5 Analysis of Inhibition of HDAC5

The potency of exemplifying compounds described herein is quantified bymeasuring the Histone Deacetylase 5 (HDAC5) enzymatic activity using theClass IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate isdeacetylated to Boc-Lys-AMC by HDAC5. Cleavage by trypsin results in therelease of the fluorophore AMC from the deacetylated substrate. Thefluorescence of the sample is directly related to the histonedeacetylase activity in the sample.

Serially Dilute the Compounds.

Serial dilutions of the compounds to be tested and control referencecompound(1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone)are made by first resuspending the lyophilized compound to a finalconcentration of 10 mM in 100% DMSO. Stocks of 60 μL aliquots of the 10mM compound in DMSO are prepared and stored at −20° C. From one stockaliquot of each compound to be tested and the reference compound, a16-point serial dilution is prepared according to Table 1 using a 125 μL16-channel Matrix multi-channel pipette.

2 μL (200×) of each diluted solution and each control (full activity:100% DMSO alone or full inhibition 1 mM) is stamped into V-bottompolypropylene 384-well compound plates using either Bravo, Janus, or a12.5 μL 16-channel Matrix multi-channel pipette. Each well with the 2 μLof the 200× stamped compound solution is diluted 1:20 by the addition of38 μL assay buffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mMKCl, and 0.9 mM MgCl₂ at pH 8.0 and equilibrated to 37° C.).

Prepare HDAC5 Catalytic Domain Enzyme (0.57 μg/mL).

The HDAC5 catalytic domain enzyme is human HDAC5 catalytic domain(GenBank Accession No. NM_(—)001015053), amino acids 657-1123 with aC-terminal His tag and can be obtained from BPS BioScience. The proteinis 51 kDa and is expressed in a baculovirus expression system. A workingsolution of enzyme is prepared from a 1.65 mg/mL stock aliquot of HDAC5catalytic domain (thawed on ice) diluted to 0.57 μg/mL with assay buffer(50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1 mM MgCl₂ at pH 8 andequilibrated to 37° C.) just prior to the addition of the enzyme to theassay.

Prepare 5× (40 μM) Boc-Lys(Tfa)-AMC Substrate.

5× (40 μM) substrate is prepared just prior to the addition to theassay. The 5× substrate is prepared by diluting the 100 mMBoc-Lys(Tfa)-AMC in DMSO solution 1:2500 by adding it drop-wise to assaybuffer (equilibrated to 37° C.) while vortexing at slow speed to preventprecipitation.

Prepare 3× (30 μM) Developer/Stop Solution.

3× (30 μM) Developer/Stop Solution is prepared just prior to addition tothe plate by diluting a stock solution of 10 mM reference compound 1:333in 25 mg/ml trypsin equilibrated to 37° C.

Assay.

5 μL of each solution of the 1:20 diluted compounds and controls fromabove is transferred to a clear bottomed, black, 384-well assay plateusing the Bravo or Janus. Using a 16-channel Matrix multi-channelpipette, 35 μL of the working solution of the HDAC5 catalytic domainenzyme (0.57 μg/mL in assay buffer) is transferred to the assay plate.The assay is then started by adding 10 μL of 5× (40 μM) substrate to theassay plates using either the Bravo, Janus or 16-channel Matrixmulti-channel pipette. The assay plate is then shaken for one minute onan orbital shaker at 900 rpm. Next, the plates are incubated for 15minutes at 37° C. The reaction is stopped by adding 25 μL of 3× (30 μM)developer/stop solution to the assay plates using either the Bravo,Janus or a 16-channel Matrix multi-channel pipette. Assay plates arethen shaken for 2 minutes on an orbital shaker at 900 rpm. Next, theassay plates are incubated at 37° C. for 1 hour in a tissue cultureincubator followed by shaking for 1 minute at the maximum rpm on anorbital shaker before reading on the EnVision. Finally, the fluorescenceis measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmerEnVision in top read mode.

Example 6 Analysis of Inhibition of HDAC7

The potency of exemplifying compounds described herein is quantified bymeasuring the Histone Deacetylase 7 (HDAC7) enzymatic activity using theClass IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate isdeacetylated to Boc-Lys-AMC by HDAC7. Cleavage by trypsin results in therelease of the fluorophore AMC from the deacetylated substrate. Thefluorescence of the sample is directly related to the histonedeacetylase activity in the sample.

Serially Dilute the Compounds.

Serial dilutions of the compounds to be tested and control referencecompound(1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone)are made by first resuspending the lyophilized compound to a finalconcentration of 10 mM in 100% DMSO. Stocks of 60 μL aliquots of the 10mM compound in DMSO are prepared and stored at −20° C. From one stockaliquot of each compound to be tested and the reference compound, a16-point serial dilution is prepared according to Table 1 using a 125 μL16-channel Matrix multi-channel pipette.

2 μL (200×) of each diluted solution and each control (full activity:100% DMSO alone or full inhibition 1 mM) is stamped into V-bottompolypropylene 384-well compound plates using either the Bravo, Janus, ora 12.5 μL 16-channel Matrix multi-channel pipette. Each well with the200× compound solution is diluted 1:20 by the addition of 38 μL assaybuffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl, and0.9 mM MgCl₂ at pH 8.0 and equilibrated to 37° C.).

Prepare HDAC7 Enzyme (71 ng/mL).

The HDAC7 enzyme is human HDAC7 (GenBank Accession No. AY302468) aminoacids 518-end with a N-terminal Glutathione S-transferase (GST) tag andcan be obtained from BPS BioScience. The protein is 78 kDa and isexpressed in a baculovirus expression system. A working solution ofenzyme is prepared from a 0.5 mg/mL stock aliquot of HDAC7 (thawed onice) diluted to 71 ng/mL with assay buffer (50 mM Tris-HCl, 137 mM NaCl,2.7 mM KCl, and 1 mM MgCl₂ at pH 8 and equilibrated to 37° C.) justprior to the addition of enzyme to the assay.

Prepare 5× (50 μM) Boc-Lys(Tfa)-AMC substrate.

5× (50 μM) substrate is prepared just prior to the addition to theassay. The 5× substrate is prepared by diluting a 100 mMBoc-Lys(Tfa)-AMC in DMSO solution 1:2000 by adding it drop-wise to assaybuffer (equilibrated to 37° C.) while vortexing at slow speed to preventprecipitation.

Prepare 3× (30 μM) Developer/Stop Solution.

3× (30 μM) Developer/Stop Solution is prepared just prior to addition tothe plate by diluting a stock solution of 10 mM reference compound 1:333in 25 mg/mL trypsin equilibrated to 37° C.

Assay.

5 μL of each solution of 1:20 diluted compound from above is transferredto a clear bottomed, black, 384-well assay plate using the Bravo orJanus. Using a 16-channel Matrix multi-channel pipette, 35 μL of theworking solution of the HDAC7 enzyme (71 ng/mL in assay buffer) istransferred to the assay plate. The assay is then started by adding 10μL of 5× (50 μM) substrate to the assay plate using either the Bravo,Janus or 16-channel Matrix multi-channel pipette. The assay plate isthen shaken for one minute on an orbital shaker at 900 rpm. Next, theplate is incubated for 15 minutes at 37° C. The reaction is then stoppedby adding 25 μL of 3× (30 μM) developer/stop solution to the assayplates using either the Bravo, Janus or a 16-channel Matrixmulti-channel pipette. The assay plate is then shaken for 2 minutes onan orbital shaker at 900 rpm. Next, the assay plate is incubated at 37°C. for 1 hour in a tissue culture incubator followed by shaking for 1minute at maximum rpm on an orbital shaker. Finally, the fluorescence ismeasured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmerEnVision in top read mode.

Example 7 Analysis of Inhibition of HDAC9

The potency of exemplifying compounds described herein is quantified bymeasuring the Histone Deacetylase 9 (HDAC9) enzymatic activity using theClass IIa selective substrate, Boc-Lys(Tfa)-AMC. The substrate isdeacetylated to Boc-Lys-AMC by HDAC9. Cleavage by trypsin results in therelease of the fluorophore AMC from the deacetylated substrate. Thefluorescence of the sample is directly related to the histonedeacetylase activity in the sample.

Serially Dilute the Compounds.

Serial dilutions of the compounds to be tested and control referencecompound(1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone)are made by first resuspending the lyophilized compound to a finalconcentration of 10 mM in 100% DMSO. Stocks of 60 μL aliquots of the 10mM compound in DMSO are prepared and stored at −20° C. From one stockaliquot of each compound to be tested and the reference compound, a16-point serial dilution is prepared according to Table 1 using a 125 μL16-channel Matrix multi-channel pipette.

2 μL (200×) of each diluted solution and each control (full activity:100% DMSO alone or full inhibition 1 mM) is stamped into V-bottompolypropylene 384-well compound plates using either the Bravo, Janus, or12.5 μL 16-channel Matrix multi-channel pipette. Each well with thestamped 200× compound solution is diluted 1:20 by the addition of 38 μLassay buffer+DMSO (10.5% DMSO, 45 mM Tris-HCl, 123 mM NaCl, 2.4 mM KCl,and 0.9 mM MgCl₂ at pH 8.0 and equilibrated to 37° C.).

Prepare HDAC9 Enzyme (0.57 μg/mL).

The HDAC9 enzyme is human HDAC9 (GenBank Accession No. NM_(—)178423)amino acids 604-1066 with a C-terminal His tag and can be obtained fromBPS BioScience. The protein is 50.7 kDa and is expressed in abaculovirus expression system. A working solution of enzyme is preparedfrom a 0.5 mg/mL stock aliquot of HDAC9 (thawed on ice) diluted to 0.57μg/mL with assay buffer (50 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, and 1mM MgCl₂ at pH 8 and equilibrated to 37° C.) just prior to the additionof enzyme to the assay.

Prepare 5× (125 μM) Boc-Lys(Tfa)-AMC Substrate.

5× (125 μM) substrate is prepared just prior to the addition to theassay. The 5× substrate is prepared by diluting a 100 mMBoc-Lys(Tfa)-AMC in DMSO solution 1:800 by adding it drop-wise to assaybuffer (equilibrated to 37° C.) while vortexing at slow speed to preventprecipitation.

Prepare 3× (30 μM) Developer/Stop Solution.

3× (30 μM) Developer/Stop Solution is prepared just prior to addition tothe plate by diluting a stock solution of 10 mM reference compound 1:333in 25 mg/mL trypsin equilibrated to 37° C.

Assay.

5 μL of each solution of 1:20 diluted compound from above is transferredto a clear bottomed, black, 384-well assay plate using the Bravo orJanus. Using a 16-channel Matrix multi-channel pipette, 35 μL of theworking solution of the HDAC9 enzyme (0.57 μg/mL in assay buffer) istransferred to the assay plate. The assay is then started by adding 10μL of 5× (125 μM) substrate to the assay plate using either the Bravo,Janus or 16-channel Matrix multi-channel pipette. The assay plate isthen shaken for one minute on an orbital shaker at 900 rpm. Next, theplate is incubated for 15 minutes at 37° C. The reaction is stopped byadding 25 μL of 3× developer/stop solution to the assay plates usingeither the Bravo, Janus or a 16-channel Matrix multi-channel pipette.The assay plate is then shaken for 2 minutes on an orbital shaker at 900rpm. Next, the assay plate is incubated at 37° C. for 1 hour in a tissueculture incubator followed by shaking for 1 minute at maximum rpm on anorbital shaker before reading on the EnVision. Finally, the fluorescenceis measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmerEnVision in top read mode.

Example 8 Analysis of Inhibition of Cellular HDAC Activity

The potency of exemplifying compounds described herein is quantified bymeasuring the cellular histone deacetylase enzymatic activity using theClass IIa selective substrate, Boc-Lys(Tfa)-AMC. After penetration inJurkat E6-1 cells, the substrate is deacetylated to Boc-Lys-AMC. Aftercell lysis and cleavage by trypsin, the fluorophore AMC is released fromthe deacetylated substrate only. The fluoresence of the sample isdirectly related to the histone deacetylase activity in the sample.

Jurkat E6.1 Cell Culture and Plating.

Jurkat E6.1 cells are cultured according to standard cell cultureprotocols in Jurkat E6.1 Growth Media (RPMI without phenol red, 10% FBS,10 mM HEPES, and 1 mM Sodium Pyruvate). Jurkat E6.1 cells are countedusing a Coulter Counter and resuspended in Jurkat E6.1 growth media at aconcentration of 75,000 cells/35 μL. 35 μL or 75,000 cells is seededinto Greiner microtitre assay plates. The plates are then incubated at37° C. and 5% CO₂ while other assay components are being prepared.

Serially Dilute HDAC Inhibitor Compounds.

Serial dilutions of the HDAC inhibitors and control reference compound(1-(5-(3-((4-(1,3,4-oxadiazol-2-yl)phenoxy)methyl)-1,2,4-oxadiazol-5-yl)thiophen-2-yl)-2,2,2-trifluoroethanone)are made by first resuspending the lyophilized compound to a finalconcentration of 10 mM in 100% DMSO. Stocks of 70 μL aliquots of the 10mM compound in DMSO are prepared and stored at −20° C. From one stockaliquot of each compound to be tested and the reference compound, a16-point serial dilution is prepared according to Table 1 using a 125 μL16-channel Matrix multi-channel pipette.

2 μL (200×) of each diluted solution and each control (full activity:100% DMSO alone or full inhibition 1 mM) is stamped into V-bottompolypropylene 384-well compound plates using either the Bravo, Janus, or12.5 μL 16-channel Matrix multi-channel pipette. Each well with the 200×compound solution is diluted 1:20 by the addition of 38 μL Jurkat assaybuffer+DMSO (9.5% DMSO, RPMI without phenol red, 0.09% FBS, 9 mM Hepes,and 0.9 mM Sodium Pyruvate equilibrated to r.t.)

Prepare 5× (500 μM) Boc-Lys(Tfa)-AMC Substrate.

5× (500 μM) substrate is prepared just prior to the addition to theassay. The 5× substrate is prepared by diluting a 100 mMBoc-Lys(Tfa)-AMC in DMSO solution 1:200 by adding it drop-wise to Jurkatassay medium (RPMI without phenol red, 0.1% FBS, 10 mM Hepes, and 1 mMSodium Pyruvate equilibrated to 37° C.) while vortexing at slow speed toprevent precipitation.

Prepare 3× Lysis Buffer.

10 mL of 3× lysis buffer is prepared with 8.8 mL of 3× stock lysisbuffer (50 mM Tris-HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 1%Nonidet P40 Substitute equilibrated to r.t.) and 1.2 mL of 3 mg/mLTrypsin equilibrated to r.t.

Assay.

5 μL of each solution of 1:20 diluted compound from above is transferredto the Greiner microtitre assay plates with 75,000 cells/well using theBravo. Cells are then incubated for 2 hours at 37° C. and 5% CO₂. Theassay is then started by adding 10 μL of 5× (500 μM) substrate to theassay plate using either the Bravo or 16-channel Matrix multi-channelpipette. The cells are then incubated for 3 hours at 37° C. and 5% CO₂.Next, 25 μL of 3× lysis buffer is added to each well using either the125 μL 16 channel pipette or the Bravo. The assay plate is thenincubated overnight (15-16 hours) at 37° C. and 5% CO₂. The followingday, the plates are shaken on an orbital shaker for 1 minute at 900 rpm.Finally the top read fluorescence (Excitation: 355 nm, Emission: 460 nm)is measured using PerkinElmer EnVision.

Example 9 Compound 6 PK Study

Following iv administration at a nominal dose of 5 mg eq./Kg Compound 6exhibited an apparent bi-phasic elimination. Therefore the clearancevalue of 6.4 L/h/Kg (>liver plasma flow) for Compound 6, is consideredto be an average of the clearance phases (data in Tables 2 and 3).

There were insufficient data to characterise the terminal eliminationhalf-life in plasma for Compound 6. Using half-life values determined inplasma or brain tissue and the volume of distribution at steady state,the clearance of the terminal phase was estimated to be 0.13 L/hr/Kg(<5% liver plasma flow) for Compound 6. Evidence of glucuronidation wasobserved during sample analysis, therefore direct conjugation of thehydroxamate is a potential clearance mechanism.

Following oral dosing (nominal 10 mg/Kg) Compound 6 was absorbed rapidly(t_(max) 0.25 h post dose) and its bioavailability was determined to begood (32%).

Compound 6 distributed into brain and muscle tissue with average tissueto plasma ratios increasing over the time course of the studies. Asevidenced by individual tissue:plasma ratios, Compound 6 showed hightissue distribution. The volume of distribution of Compound 6 wascalculated as being 1.1 L/Kg.

Example 10 Compound 8 PK Study

Higher brain penetration and volume of distribution were observed withCompound 8 (data shown in Table 2) compared to other compounds.

Compound 8 Dose Escalation PK Study

Compound 8 was progressed into a dose escalation study (POadministration to C57Bl6 mice, 30 and 100 mg eq./kg). The concentrationsof Compound 8 determined in plasma, brain and muscle from this study andthe previous study where Compound 8 was administered PO at 10 mg/kg areshown in Tables 2 and 3.

Plasma, brain and muscle concentrations of Compound 8 following POadministration at 10 mg/kg, 30 mg/kg and 100 mg/kg are shown in FIG. 1.

Following oral doses (30 and 100 mg eq./kg), the compound was absorbedrapidly with C_(max) occurring at 0.5 hours post dose. The plasmaAUC_(0-last) appeared linear with increasing dose levels with a dosenormalised value of approximately 300 nM·hr·kg/mg. However, C_(max) didnot show a linear increase suggesting that dissolution may be decreasingthe rate of absorption, but not its extent, with increasing dose.Bioavailability was good at ca. 35% for both dose levels.

Evidence of glucuronidation was observed during sample analysis,therefore direct conjugation of the hydroxamate is a potential clearancemechanism for this compound.

Compound 8 showed good distribution into brain and muscle tissue.Terminal half-lives could not be determined accurately but wereestimated to be 4.8 h (PO 30 mg/kg) and 3.4 h (PO 100 mg/kg) hours inbrain and 5.6 h (PO 30 mg/kg) and 4.1 h (PO 100 mg/kg) in muscle.

When compared to the 30 mg/Kg PO data, dose normalized brain and muscleAUCs decreased by approximately 20% when Compound 8 was dosed at 100mg/kg suggesting that brain permeability and penetration into the muscleis approaching saturation, similar to that seen for Compound 6.Additionally, t_(max) was shown to be later (1 h) at the higher doselevel, further indicating that the rate of absorption of Compound 8 isdecreasing with increased dose.

Compound 8: absorption, distribution, metabolism, and excretionprofiling, thermodynamic solubility and dose formulation studies

Compound 8 was shown to be highly bound to plasma proteins (3% unbound)and brain tissue (0.5% unbound) in contrast to the low plasma proteinand brain tissue binding determined for Compound 6 (20% and 10% unboundrespectively).

The thermodynamic solubility of Compound 8 was highest at pH 9 butdetermined to be moderate at all pHs (0.012-0.035 mg/mL).

Example 11 Hepatic Microsomal Stability (Half-Life)

Incubations of test compounds (1 μM initial concentration, n=2) werecarried out with pooled hepatic microsomes (0.25 mg protein/mL in 0.1 Mphosphate buffer pH7.4). NADPH (1 mM) was added to initiate thereactions. The incubations were performed at 37° C. Samples (100 μL)were taken from the incubation at 0, 5, 10, 20 and 40 min and added to100 μL of acetonitrile containing carbamazepine as analytical internalstandard, to terminate the reaction. Samples were centrifuged and thesupernatant fractions analysed by LC-MS/MS. The instrument responses(i.e. chromatographic peak heights), normalized by internal standardresponse were referenced to the zero time-point samples (as 100%) inorder to determine the percentage of compound remaining.

Ln plots of the % remaining, for each compound, were used to determinethe half-life for the microsomal incubations.

Half-life values were calculated from the relationship

T½(min)=−0.693/λ

where λ was the slope of the Ln concentration vs time curve.

The in vitro intrinsic clearance, CL_(int) (μL/min/mg microsomalprotein), was calculated using the following formula:

Cl _(int)=0.693×1/T _(1/2)(min)×incubation volume(μL)/mg of microsomalprotein

The in vitro intrinsic clearance, Cl_(int) (mL/min/kg), was calculatedand scaled to hepatic extraction ratios using the following scalingparameters and formulae.

Parameters

Value Parameter Human Mouse Rat Microsomal protein concentration 0.250.25 0.25 in incubation (mg/mL) microsomes/g liver (mg) 52 52 45 liverweight/kg body weight (g) 25 45 40 hepatic plasma flow (mL/min/kg) 11.750 30

Formulae

Cl _(int)(tissue clearance)mL/min/kg=[0.693/t ^(1/2)(min)]×[1/microsomalprotein concentration mg/mL]×[mg microsomes/g liver]×[g liver/kg bodyweight]

Cl _(int)(hepatic clearance)mL/min/kg=hepatic plasma flow×Cl_(int)/(hepatic plasma flow+Cl_(int))

Hepatic extraction ratio(Eh)=Cl _(int)(hepaticclearance)mL/min/kg/hepatic plasma flow(mL/min/kg)

Permeability and Effective Efflux Ratio in MDCK-MDR1

The MDR1-MDCKII and wild type MDCKII cell lines were cultured inaccordance with the guidelines provided by Solvo Biotechnology. Bothwild-type MDCK and MDR1-MDCK cells were seeded at a cell density of2.3×10⁵ cells/well into 24-well Transwell plates and cultured for threedays to form monolayers. Test compound was loaded into the donorcompartments of the Transwell plate (24-well) bearing MDR1-MDCK or wildtype MDCK monolayers. Test compound was added to either the apical orbasolateral chambers of the Transwell plate assembly at a concentrationof 10 μM in Hanks' Balanced Salt Solution containing 25 mM HEPES (pH7.4). Lucifer Yellow was added to the apical buffer in all wells and itspermeation monitored to assess integrity of the cell layer. As LuciferYellow (LY) cannot freely permeate lipophilic barriers, a high degree ofLY transport indicates poor integrity of the cell layer and wells withLY permeability above 100 nm/s are rejected.

After 1 h incubation at 37° C., aliquots were taken from both chambersand added to acetonitrile containing analytical internal standard(carbamazepine) in a 96-well plate. Concentrations of compound in thesamples were measured by LC-MS/MS. Concentrations of LY in the sampleswere measured using a fluorescence plate reader.

The apparent permeability (P_(app)) values of test compound weredetermined for both the apical to basal (A>B) and basal to apical (B>A)permeation and the efflux ratio (B>A:A>B) determined in both the wildtype MDCK and MDR1-MDCK cells.

Apparent permeability (P_(app)) values were calculated from therelationship:

${P_{app}\left( {{cm}\text{/}\sec \times 10^{- 6}} \right)} = {\left\lbrack \frac{\begin{matrix}{{compound}_{{acceptor}\mspace{14mu} {final}} \times} \\{V_{acceptor} \times V_{donor}}\end{matrix}}{\begin{matrix}{{compound}_{{donor}\mspace{14mu} {initial}} \times V_{donor} \times} \\{T_{inc} \times {surface}\mspace{14mu} {area}}\end{matrix}} \right\rbrack \times 10^{6}}$

Where V=chamber volume and T_(inc)=incubation time in seconds.

Donor=Chamber of Transwell to which compound is dosed: apical for A>Bexperiments and basal for B>A experiments.

Acceptor=Chamber of Transwell in to which permeation of compound ismeasured: basal for A>B experiments and apical for B>A experiments.

The Efflux ratios, as an indication of active efflux from the apicalcell surface, were calculated using the ratio of P_(app) B>A/P_(app)A>B.

The effective efflux ratio was also determined from the ratio observedin MDR1-MDCK cells relative to the ratio observed in wild-type cells.Known substrates for human MDR1 typically display effective effluxratios of greater than two.

The data are shown in Tables 2 and 3.

TABLE 2

cat. HDAC4 IC₅₀ (μM) 0.049 0.044 HDAC4 (bioch) IC₅₀ (μM) 0.04 0.06 HDAC4(cell) IC₅₀ (μM) 0.11 0.07 cat. HDAC5 IC₅₀ (μM) 0.023 0.060 cat. HDAC7IC₅₀ (μM) 0.026 0.031 cat. HDAC9 IC₅₀ (μM) 0.040 0.050 cat. HDAC3 IC₅₀(μM) 25.1 25.8 cat. HDAC8 IC₅₀ (μM) 6.7 9.1 cat. HDAC6 IC₅₀ (μM) 3.0 NTBoc-Lys(Tfa) IC₅₀ (μM) 0.12 0.059 Boc-Lys(Ac) IC₅₀ (μM) >50 >50 M_(w)337 391 tPSA [Å²] 75 75 AlogP 2.3 3.4 kinetic solubility 0.059 0.056 (mgeq./mL) effective efflux ratio (MDR1- 3.0 1.2 MDCK/wt-MDCK) MDCKPAPP_A_B (nm/s) 280 294 human liver m/somes Cl_(int) <36 <36 ml/min/KgBW mouse liver m/somes Cl_(int) 109 <76 mL/min/Kg BW CYP P450 inhibition(μM) >50 all isozymes tested >50 all isozymes tested except 3A4, >35Plasma protein binding (% 20 3 unbound) Brain tissue binding (% 9.9 0.4unbound) Plasma Cmax_(norm) (nM) po, 220 280 Plasma AUC_(norm) (nM*h) iv460 830 Plasma Clearance (L/h/kg) 6.4 3.1 Volume_(ss) (L/kg) 1.1 7.3 ivt_(1/2) (h) Pl NC Pl 3.4 Br 5.7 Br 2.0 Mu 8.4 Mu NC Oral Bioavailability(%) 32 44 Br:Pl ratio iv 0.2-4.5:1 1.5-4.9:1 Mu:Pl ratio iv 0.9-12:1 1.0-2.7:1 Plasma Cmax_(norm) (nM) po 200, 160 132, 70 Plasma AUC_(norm)(nM*h) iv 930 — Plasma AUC_(norm) (nM*h) po 250, 250 300, 290 PlasmaClearance (L/h/kg) 3.2 — Volume_(ss) (L/kg) 1.04 — iv t_(1/2) (h) Pl 2.2— Br 5.4 Mu 7.0 po t_(1/2) (h) Pl 1.9, 1.2 NC Br 4.4, 3.6 Mu 8.2, 8.8Oral Bioavailability (%) 27, 27 36, 35 Tissue:Plasma Br:Pl 0.1-3.7:1,Mu:Pl 0.5-23:1 Br:Pl 0.2-2.2:1, Mu:Pl 0.4-1.9:1 Key: NT  Not tested NC Not calculated due to insufficient data points NA  Not applicable PKparameters due to route —   Study not performed Br   Brain Pl   PlasmaMu  Muscle AlogP and tPSA calculated with Pipeline Pilot 8.5, AccelrysSoftware Inc.

TABLE 3 Compound 6 Compound 8

IC₅₀ μM (bioch) 0.04 0.06 IC₅₀ μM (cell) 0.11 0.07 AlogP 2.3 3.4 MLM 65,109 76 (mL/min/kg BW) MDCK EER 3.0 1.2 Papp A-B (cm/s x 10⁶) 280 294 IVPK Plasma AUC_(norm) 463 830 nM*hr kg/mg Clearance (CL_(p)) 6.4 3.1Volume of Distribution 1.1 7.3 at Steady State (Vd_(ss)) L/Kg Half-lifehour NC 3.4 Brain AUC_(norm) 274 1600 nM*hr kg/mg Clearance (CL_(p)) 2943100 Half-life hour 5.7 2.0 Brain-plasma 0.19-4.5 1.5-4.9 MuscleAUC_(norm) 900 800 nM*hr kg/mg Clearance (CL_(p)) 6915 6000 Half-lifehour 8.4 NC Brain-plasma 0.85-9.7 0.98-2.7 Oral PK Plasma AUC_(norm) 149360 nM hr kg/mg Bioavaiability % 32 43 Observed 215 280 C_(maxNorm) nMkg/mg Half-life hour NC NC Brain AUC_(norm) 52 480 nM*hr kg/mg Clearance(CL_(p)) 23 310 Half-life hour 3.3 5.1 Brain-plasma 0.04-0.7 0.35-2.6Muscle AUC_(norm) 192 290 nM*hr kg/mg Clearance (CL_(p)) 901 3000Half-life hour 5.5 NC Muscle-plasma 0.12-4.1 0.56-3.1 Comparisoncompounds

IC₅₀ μM (bioch) 0.07 0.09 IC₅₀ μM (cell) 0.64 0.55 AlogP 2.4 2.1 MLM 9377 (mL/min/kg BW) MDCK EER 3.2 4.9 Papp A-B (cm/s x 10⁶) 234 175 IV PKPlasma AUC_(norm) 409 516 nM*hr kg/mg Clearance (CL_(p)) 7.4 6.4 Volumeof Distribution 3.1 2.6 at Steady State (Vd_(ss)) L/Kg Half-life hour1.8 1.8 Brain AUC_(norm) 511 162 nM*hr kg/mg Clearance (CL_(p)) 666 172Half-life hour 3.3 2.2 Brain-plasma 0.54-8.9 0.12-2 Muscle AUC_(norm)1048 835 nM*hr kg/mg Clearance (CL_(p)) 2378 1689 Half-life hour 4.8 4.0Brain-plasma 1.9-21 1.1-8.9 Oral PK Plasma AUC_(norm) 109 179 nM hrkg/mg Bioavaiability % 27 35 Observed 95 257 C_(maxNorm) nM kg/mgHalf-life hour NC 1.9 Brain AUC_(norm) 151 58 nM*hr kg/mg Clearance(CL_(p)) 63 50 Half-life hour NC 2.7 Brain-plasma 0.67-3.4 0.15-1.4Muscle AUC_(norm) 260 306 nM*hr kg/mg Clearance (CL_(p)) 145 264Half-life hour NC 4.0 Muscle-plasma 1.5-5.2 1.0-8.0 NC: Not calculated,insufficient data points AlogP calculated with Pipeline Pilot 8.5,Accelrys Software Inc.

TABLE 4 Fold Selectivity (bioch)

HDAC4 (μM) 0.06 0.09 HDAC5:HDAC4 0.9 0.3 HDAC7:HDAC4 0.5 0.8 HDAC9:HDAC40.8 1 HDAC3:HDAC4 400 320 HDAC8:HDAC4 140 60 HDAC6:HDAC4 ND 50

While some embodiments have been shown and described, variousmodifications and substitutions may be made thereto without departingfrom the spirit and scope of the invention. For example, for claimconstruction purposes, it is not intended that the claims set forthhereinafter be construed in any way narrower than the literal languagethereof, and it is thus not intended that exemplary embodiments from thespecification be read into the claims. Accordingly, it is to beunderstood that the present invention has been described by way ofillustration and not limitations on the scope of the claims.

1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein R is pyrimidinesubstituted with 1 or 2 groups independently chosen from halo, C₁-C₄alkyl, and C₁-C₄ haloalkyl; each R¹ is independently chosen from halo,C₁-C₄ alkyl, and C₁-C₄ haloalkyl; and m is 1, 2 or
 3. 2. The compound ofclaim 1, or a pharmaceutically acceptable salt thereof, wherein R is

wherein each R² is independently chosen from halo, C₁-C₄ alkyl, andC₁-C₄ haloalkyl; n is 1 or 2; and

represents the point of connection to the rest of the molecule.
 3. Thecompound of claim 2, or a pharmaceutically acceptable salt thereof,wherein n is
 1. 4. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R is


5. The compound of claim 2, or a pharmaceutically acceptable saltthereof, wherein each R² is independently C₁-C₄ alkyl or C₁-C₄haloalkyl.
 6. The compound of claim 5, or a pharmaceutically acceptablesalt thereof, wherein each R² is independently methyl ortrifluoromethyl.
 7. The compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein R is


8. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein m is
 1. 9. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein at least one R¹ ishalo.
 10. The compound of claim 9, or a pharmaceutically acceptable saltthereof, wherein at least one R¹ is fluoro.
 11. The compound of claim10, or a pharmaceutically acceptable salt thereof, wherein m is 1, andR¹ is 2-fluoro.
 12. A compound, which is2-(2-fluorophenyl)-N-hydroxy-2-(4-(2-methylpyrimidin-5-yl)phenyl)acetamide,of the formula:

or2-(2-fluorophenyl)-N-hydroxy-2-(4-(5-(trifluoromethyl)pyrimidin-2-yl)phenyl)acetamide,of the formula:

or a pharmaceutically acceptable salt thereof.
 13. A pharmaceuticallyacceptable composition comprising a pharmaceutically acceptable carrierand at least one compound of claim 1, or a pharmaceutically acceptablesalt thereof.
 14. A pharmaceutical composition of claim 13, wherein thecomposition is formulated in a form chosen from tablets, capsules,powders, liquids, suspensions, suppositories, and aerosols.
 15. A methodfor treating a condition or disorder mediated by at least one histonedeacetylase in a patient in need of such a treatment which methodcomprises administering to the patient a therapeutically effectiveamount of at least one compound of claim 1, or a pharmaceuticallyacceptable salt thereof.
 16. A method for treating a condition ordisorder responsive to inhibition of at least one histone deacetylase ina patient in need of such a treatment which method comprisesadministering to the patient an effective amount of at least onecompound of claim 1, or a pharmaceutically acceptable salt thereof. 17.A method for inhibiting at least one histone deacetylase which methodcomprises contacting the histone deacetylase with an effective amount ofat least one compound of claim 1, or a pharmaceutically acceptable saltthereof.
 18. The method of claim 15, wherein the at least one histonedeacetylase is chosen from HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8,HDAC-9, HDAC-10 and HDAC-11.
 19. The method of claim 18, wherein the atleast one histone deacetylase is chosen from HDAC-4, HDAC-5, HDAC-7, andHDAC-9.
 20. The method of claim 19, wherein the at least one histonedeacetylase is HDAC-4.
 21. The method of claim 15, wherein saidcondition or disorder is chosen from a cell proliferative disease,diabetes, inflammation, cardiac disease, stroke, epilepsy, depression,immunological disease and viral or fungal infection.
 22. The method ofclaim 15, wherein said condition or disorder involves aneurodegenerative pathology.
 23. The method of claim 22, wherein thecondition or disorder is Huntington's disease.